Compositions of PD-1 antagonists and methods of use

ABSTRACT

Methods of treating cancer and infectious diseases utilizing a treatment regimen comprising administering a compound that reduces inhibitory signal transduction in T cells, in combination with a potentiating agent, such as cyclophosphamide, to produce potent T cell mediated responses, are described. Compositions comprising the PD-1 antagonists and potentiating agents useful in the methods of the invention are also disclosed.

This application is a divisional of U.S. Ser. No. 12/547,129 filed Aug.25, 2009, entitled “Compositions of PD-1 Antagonists and Methods ofUse”, by Solomon Langermann and Linda Liu, which claims priority to U.S.Ser. No. 61/211,697 filed Apr. 2, 2009, U.S. Ser. No. 61/091,694 filedAug. 25, 2008, U.S. Ser. No. 61/091,709 filed Aug. 25, 2008, and U.S.Ser. No. 61/091,705 filed Aug. 25, 2008, all of which are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to therapeutic compositions containing acompound that prevents inhibitory signal transduction on T cells incombination with potentiating agents and the use of said componentstogether or separately for the induction of T cell responses valuable indisease therapy.

BACKGROUND OF THE INVENTION

The response of T lymphocytes to disease states, such as infection andchronic diseases like cancer, is complicated and involves intercellularinteractions and the production of soluble mediators (called cytokinesor lymphokines). Activation of T cells normally depends on anantigen-specific signal following contact of the T cell receptor (TCR)with an antigenic peptide presented via the major histocompatibilitycomplex (MHC) while the extent of this reaction is controlled bypositive and negative antigen-independent signals eminating from avariety of co-stimulatory molecules. The latter are commonly members ofthe CD28/B7 family. Conversely, Programmed Death-1 (PD-1) is a member ofthe CD28 family of receptors that delivers a negative immune responsewhen induced on T cells. Contact between PD-1 and one of its ligands(B7-H1 or B7-DC) induces an inhibitory response that decreases T cellmultiplication and/or the strength and/or duration of a T cell response.

Thus, the T lymphocyte response is regulated by various factors,including cell surface molecules that act as receptors, where the latterinclude both the TCR complex as well as other surface molecules.

In sum, an antigen specific T cell response is mediated by twosignals: 1) engagement of the TCR with antigenic peptide presented inthe context of HC (signal 1), and 2) a second antigen-independent signaldelivered by contact between different receptor/ligand pairs (signal 2).This “second signal” is critical in determining the type of T cellresponse (activation vs tolerance) as well as the strength and durationof that response, and is regulated by both positive and negative signalsfrom costimulatory molecules, such as the B7 family of proteins.

The most extensively characterized T cell costimulatory pathway isB7-CD28, in which B7-1 (CD80) and B7-2 (CD86) each can engage thestimulatory CD28 receptor and the inhibitory CTLA-4 (CD152) receptor. Inconjunction with signaling through the T cell receptor, CD28 ligationincreases antigen-specific proliferation of T cells, enhances productionof cytokines, stimulates differentiation and effector function, andpromotes survival of T cells (Lenshow, et al., Annu. Rev. Immunol.,14:233-258 (1996); Chambers and Allison, Curr, Opin. Immunol., 9:396-404(1997); and Rathmell and Thompson, Annu. Rev. Immunol., 17:781-828(1999)). In contrast, signaling through CTLA-4 is thought to deliver anegative signal that inhibits T cell proliferation, IL-2 production, andcell cycle progression (Krummel and Allison, J. Exp. Med., 183:2533-2540(1996); and Walunas, et al., J. Exp. Med., 183:2541-2550 (1996)). Othermembers of the B7 family of costimulatory molecules include B7-H1 (Dong,et al, Nature Med., 5:1365-1369 (1999); and Freeman, et al., J. Exp.Med., 192:1-9 (2000)), B7-DC (Tseng, et al., J. Exp. Med., 193:839-846(2001); and Latchman, et al., Nature Immunol., 2:261-268 (2001)), B7-H2(Wang, et al., Blood, 96:2808-2813 (2000); Swallow, et al., Immunity,11:423-432 (1999); and Yoshinaga, et al., Nature, 402:827-832 (1999)),B7-H3 (Chapoval, et al., Nature Immunol., 2:269-274 (2001)) and B7-H4(Choi, et al., J. Immunol., 171:4650-4654 (2003); Sica, et al.,Immunity, 18:849-861 (2003); Prasad, et al., Immunity, 18:863-873(2003); and Zang, et al., Proc. Natl. Acad. Sci. U.S.A., 100:10388-10392(2003)). B7-H5 (described in WO 2006/012232) is a newly discoveredmember of the B7 family.

B7 family molecules have a membrane proximal IgC (constant) domain and amembrane distal IgV (variable) domain. The CD28-like family of receptorsfor these ligands share a common extracellular IgV-like domain.Interactions of receptor-ligand pairs are mediated predominantly throughresidues in the IgV domains of the ligands and receptors (Schwartz, etal., Nature Immunol., 3:427-434 (2002)). In general, IgV domains aredescribed as having two sheets that each contains a layer of β-strands(Williams and Barclay, Annu. Rev. Immunol., 6:381-405 (1988)). The frontand back sheets of CTLA-4 contain strands A′GFC′C and ABEDC,respectively (Ostrov, et al., Science, 290:816-819 (2000)), whereas thefront and back sheets of the B7 IgV domains are composed of strandsAGFCC′C″ and BED, respectively (Schwartz, et al., Nature, 410:604-608(2001); Stamper, et al., Nature, 410:608-611 (2001); and Ikemizu, etal., Immunity, 12:51-60 (2000)). Crystallographic analysis revealed thatthe CTLA-4/B7 binding interface is dominated by the interaction of theCDR3-analogous loop from CTLA-4, composed of a MYPPPY motif, with asurface on B7 formed predominately by the G, F, C, C′ and C″ strands(Schwartz, et al., Nature, 410:604-608 (2001); and Stamper, et al.,Nature, 410:608-611 (2001)). Data from amino acid homologies, mutation,and computer modeling provide support for the concept that this motifalso is a major B7-binding site for CD28 (Bajorath, et al., J. Mol.Graph. Model., 15:135-139 (1997)). Although the MYPPPY motif is notconserved in ICOS, the receptor for B7-H2, studies have indicated that arelated motif having the sequence FDPPPF and located at the analogousposition is a major determinant for binding of ICOS to B7-H2 (Wand, etal., J. Exp. Med., 195:1033-1041 (2002)).

B7-DC (also called PD-L2 or CD273) is a relatively new member of the B7family, and has an amino acid sequence that is about 34% identical toB7-H1 (also called PD-L1). Human and mouse B7-DC orthologues share about70% amino acid identity. While B7-H1 and B7-DC transcripts are found invarious tissues (Dong, et al., Nature Med., 5:1365-1369 (1999);Latchman, et al., Nature Immunol., 2:261-268 (2001); and Tamura, Blood,97:1809-1816 (2001)), the expression profiles of the proteins are quitedistinct. B7-H1 is broadly expressed on a wide variety of tissue andcell types, while B7-DC expression is predominantly restricted toactivated dendritic cells (DC) and macrophages.

It has been shown that both B7-H1 and B7-DC bind to PD-1 (Freeman, etal., J. Exp. Med., 192:1027-1034 (2000)), a distant member of the CD28family with an immunoreceptor tyrosine-based inhibitory motif (ITIM) inits cytoplasmic domain (Ishida, et al., EMBO J., 11:3887-3895 (1992)).PD-1, a member of the CD28 family of receptors, is inducibly expressedon activated T cells, B cells, natural killer (NK) cells, monocytes, DC,and macrophages (Keir, et al Curr. Opin. Immunol. 19:309-314 (2007)).

The primary result of PD-1 ligation by its ligands is to inhibitsignaling downstream of the T cell Receptor (TCR). Therefore, signaltransduction via PD-1 usually provides a suppressive or inhibitorysignal to the T cell that results in decreased T cell proliferation orother reduction in T cell activation. B7-H1 is the predominant PD-1ligand causing inhibitory signal transduction in T cells. The presentinvention solves the problem of undesired T cell inhibition by providingagents that bind to PD-1 and thus prevent inhibitory signaltransduction, or else bind to ligands of PD-1 such as B7-H1, therebypreventing the ligand from binding to PD-1 to deliver an inhibitorysignal. In either case, T cell responses, such as T cell proliferationor activation, are stimulated.

B7-H1 is the predominant PD-1 ligand, likely due to its broaderdistribution and higher expression levels. PD-1 inhibition occurs onlywhen PD-1 and TCR are ligated in close proximity to each other, in thecontext of the immune synapse. PD-1 and its ligands have been the topicof several review articles.

B7-H1 is also over expressed in many cancers (including breast cancer,colon cancer, esophageal cancer, gastric cancer, glioma, leukemia, lungcancer, melanoma, multiple myeloma, ovarian cancer, pancreatic cancer,renal cell carcinoma, and urothelial cancer), and has been linked topoor prognosis. B7-H1 is expressed by many tumor cell lines, especiallyfollowing stimulation with interferon gamma (IFN-γ), and is alsoupregulated on tumor infiltrating myeloid derived suppressor cells(MDSC). For example, PD-1 is up-regulated on tumor specific CD8 T cellsand is associated with functional impairment, energy, exhaustion, andapoptosis. PD-1 upregulation has also been associated with dysfunctionaland/or suppressive phenotypes on additional cell types, such asregulatory T cells (Treg) and natural killer T (NKT) cells.

The present invention makes use of such molecular functions by providingtreatment regimens for treating diseases through increased T cellactivity, especially cancer and infectious diseases.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method of increasing Tcell responses, for example, to an antigen, in a mammal in need of suchincrease, comprising administering to said mammal a compound thatreduces inhibitory signal transduction in immune cells, especially Tcells, and a potentiating agent, wherein said treatment regimen iseffective to increase the T cell response of said mammal.

Compounds useful in the treatment regimen of the invention include thosethat bind to and block PD-1 receptors on T cells without triggeringinhibitory signal transduction, compounds that bind to PD-1 ligands toprevent their binding to PD-1, compounds that do both and compounds thatprevent expression of genes that encode either PD-1 or natural ligandsof PD-1. Such compounds are referred to herein as “PD-1 antagonists.”Compounds that bind to natural ligands of PD-1 include PD-1 itself, aswell as active fragments of PD-1, and in the case of the B7-H1 ligand,B7.1 proteins and fragments. Such antagonists include proteins,antibodies, anti-sense molecules and small organics. In a preferredembodiment, said T cell response is greater than that produced by eitherof said PD-1 antagonist or said potentiating agent when either isadministered without the other.

In another embodiment, compounds useful in the methods of the inventionare those that bind to T cell surface molecules such as CTLA4 to preventthe inhibitory signals triggered by binding of natural ligands thereofor that bind to said natural ligands. Such antagonists include proteins,antibodies, anti-sense molecules and small organics.

In a general embodiment, compounds useful in treatment regimens andcompositions of the present invention include those that bind to PD-1without triggering, inducing, increasing, facilitating and/or permittingco-ligation of PD-1 with TCR.

Preferred compounds that prevent inhibitory signal transduction throughPD-1 and thus act as PD-1 antagonists include, but are not limited to,B7-DC polypeptides, especially soluble portions of these, includingactive fragments of these, variants and homologs of these, as well asfusion proteins incorporating any of the foregoing, that bind to PD-1without triggering inhibitory signal transduction. In preferredembodiments, B7-DC comprises the amino acid sequence of SEQ ID NO: 1, 2,3 or 4. Preferred such compounds are those incorporating the solubledomain of B7-DC (i.e., without transmembrane sequence). Suitablefragments of B7-DC polypeptides include fragments containing the IgVand/or IgC domains or fragments containing only the IgV domain, with thelatter being a preferred embodiment, with amino acids 20-121 of SEQ IDNO: 1 being a preferred example of an IgV domain.

Preferred PD-1 antagonists also include, but are not limited to, activefragments of natural ligands of PD-1, such as B7-H1 polypeptides(disclosed in U.S. Pat. No. 6,803,192, incorporated by reference hereinin its entirety), especially soluble portions of these, includingvariants and homologs of these, as well as fusion proteins incorporatingany of the foregoing, that bind to PD-1 without triggering inhibitorysignal transduction.

Preferred compounds of the invention also include, but are not limitedto, compounds, including active fragments, variants and homologs, thatbind to natural ligands of PD-1, such as fragments of B7-1 that bind toB7-H1, as well as fusion proteins incorporating any of the foregoing,that bind to ligands of PD-1 to prevent the latter from binding to PD-1to trigger inhibitory signal transduction.

In another embodiment, the compositions and methods of use thereof,include a combination of a PD-1 receptor antagonist that binds to andblocks the PD-1 receptor, and a separate PD-1 receptor antagonist thatbinds to and blocks PD-1 receptor ligands. Another embodiment of thepresent invention provides PD-1 receptor antagonists that bind to thePD-1 receptor without triggering inhibitory signal transduction throughthe PD-1 receptor and also have the ability to bind and antagonize PD-1receptor ligands, such as B7-H1, that would otherwise trigger inhibitorysignal transduction through the PD-1 receptor. Other contemplated PD-1receptor antagonists include bi-specific antibodies that can bind boththe PD-1 receptor and PD-1 receptor ligands.

Preferred embodiments of compounds useful in the present invention alsoinclude antibodies that bind to PD-1 or CTLA4, thereby reducing, orabolishing, inhibitory signal transduction mediated by these sources.

Preferred compounds for use in the methods of the invention alsoinclude, but are not limited to, active fragments of ligands of CTLA4(such as B7-1 and B7-2) that bind to CTLA4 to reduce subsequentinhibitory signals yet do not bind to CD28 or otherwise inhibit positivesignal transduction by CD28.

Preferred compounds that prevent inhibitory signal transduction throughPD-1 and thus act as PD-1 antagonists include, but are not limited to,B7-DC antagonists, especially soluble portions of these, includingactive fragments of these, variants and homologs of these, as well asfusion proteins incorporating any of the foregoing, that bind to B7-DC.

In one embodiment, B7-DC polypeptides, fragments or variants thereof arecoupled to other polypeptides to form fusion proteins that antagonizethe PD-1 receptor by binding to the PD-1 receptor without causinginhibitory signal transduction through PD-1, thereby reducing, orinterfering with, ligand binding to PD-1, particularly B7-H1 binding,and thereby interfering with inhibitory signal transduction through thePD-1 receptor. Examples of such fusion proteins are polypeptidescomprising the amino acid sequence of SEQ ID NO: 9, 10, 12 or 13, aswell as homologs thereof. In one preferred embodiment, all or a portionof the extracellular domain (ECD) of B7-DC is part of a fusion proteinwherein it is linked to a second polypeptide containing an Fc portion ofan immunoglobulin. A preferred example of this is B7-DC-Ig, especiallywhere this structure is part of a homodimer wherein two B7-DC-Igmolecules are linked to each other, such as by a disulfide linkage.

In specific embodiments, fragments useful in the compounds of theinvention consist of at least 10, 15, 25, 50, 75, 100, 150, 200 or morecontiguous amino acids of a polypeptide having the desired antagonistactivity. Such fragments are also commonly part of fusion proteins foruse in the invention.

In another aspect, the present invention relates to a method ofincreasing T cell responses in a mammal in need thereof, comprisingadministering to said mammal an effective treatment regimen comprisingan anti-PD-1 antibody and a potentiating agent, wherein said treatmentregimen is effective to increase the T cell response of said mammal.

In another aspect, the present invention relates to a method ofincreasing T cell responses in a mammal in need thereof, comprisingadministering to said mammal an effective treatment regimen comprisingan immunomodulator, and a potentiating agent, wherein said treatmentregimen is effective to increase the T cell response of said mammal.Such immunomodulators include molecules that antagonize other CD28family receptors (such as CTLA4) that inhibit T cell responses. Apreferred embodiment uses an anti-CTLA4 antibody and a potentiatingagent. Additional immunomodulators include: molecules that agonize CD28family receptors (such as CD28 and ICOS) that activate T cell responses;molecules that antagonize B7 family ligands (such as B7-H1, B7-DC,B7-H4) that inhibit T cell responses; and molecules that agonize B7family ligands (such as B7.1 and B7.2) that activate T cell responses.

In additional embodiments of any of the methods of the invention, thetreatment regimen of a PD-1 antagonist compound and a potentiating agentfurther comprises at least one additional therapeutic agent. Additionaltherapeutic agents contemplated include immunomodulatory agents.Exemplary immunomodulating agents for such methods include anti-PD-1 andanti-CTLA4 antibodies.

In one embodiment, the potentiating agent is selected fromcyclophosphamide and analogs of cyclophosphamide, Sunitinib (Sutent),anti-TGFβ and Imatinib (Gleevac), a mitosis inhibitor, such aspaclitaxel, an aromatase inhibitor, such as letrozole, an A2a adenosinereceptor (A2AR) antagonist, an angiogenesis inhibitor, anthracyclines,oxaliplatin, doxorubicin, TLR4 antagonists, and IL-18 antagonists. Someof these agents reduce the number of Tregs (i.e., regulatory Tlymphocytes or T-regs) within the tumor microenvironment.

In another embodiment, the methods and/or compositions of the inventionspecifically contemplate use of any suitable adjuvant as part of saidmethod and/or composition.

In accordance with the invention, T cells can be contacted with PD-1receptor antagonist and/or compositions thereof containing apotentiating agent in vitro, ex vivo or in viva. Contacting T cellsusing PD-1 receptor antagonists and/or compositions thereof containing apotentiating agent can occur before, during or after activation of the Tcell.

In a specific embodiment, a molecule that prevents or reduces inhibitorysignal transduction through PD-1 and the potentiating agent areadministered at different times, such as where the potentiating agent isadministered prior to administering the PD-1 antagonist. Suchadministration may be in conjunction with an additional therapeuticagent.

In specific embodiments of any of the methods of the invention, thetreatment regimen includes administration of the potentiating agent atleast 1 hour, or at least 2 hours, or at least 3 hours, or at least 5hours, or at least 10 hours, or at least 15 hours, or at least 20 hours,or at least 24 hours, or at least 30 hours or even longer beforeadministering any or all of the PD-1 antagonist, the anti-PD-1 antibody,the anti-CTLA4 antibody, and/or additional therapeutic agents.Administration of the potentiating agent may also occur afteradministering any or all of the PD-1 antagonist, the anti-PD-1 antibody,the anti-CTLA4 antibody and/or additional therapeutic agents, such as nomore than 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20hours, 24 hours, or even up to 30 hours after administering a PD-1antagonist, or may occur in conjunction with administering the PD-1antagonist.

The increased T cell response achieved as a result of the methods of theinvention is sufficient to treat a disease, including one or more ofcancer, viral infection, bacterial infection and parasitic infection.Where the disease is cancer, such cancer is any one or more of bladder,brain, breast, cervical, colo-rectal, esophageal, kidney, liver, lung,nasopharangeal, pancreatic, prostate, skin, stomach, uterine, ovarian,testicular, or hematologic cancer.

In another aspect, the present invention includes compositions of theantagonists used in the methods of the invention, in a pharmaceuticallyacceptable carrier and wherein said PD-1 binding molecule and saidpotentiating agent are each present in an amount effective to produceincreased T cell stimulation.

In one preferred embodiment, the invention includes medical kitscomprising containers holding one or more of the agents for use in theinvention together with pharmaceutical carriers for dilution thereof andinstructions for administration. In addition, both of said PD-1 receptorantagonist and potentiating agent may be present as components in asingle container, in a pharmaceutically acceptable carrier, when saidcomponents are to be administered at the same time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that B7-DC-Ig binds to PD-1. Labeled B7-DC-Ig was incubatedat various concentrations with a CHO cell line constitutively expressingPD-1 or parent CHO cells that do not express PD-1. Binding was analyzedby flow cytometry. The median fluorescence intensity (MFI) of B7-DC-Ig(y-axis) is shown as a function of the concentration of probe (x-axis).B7-DC-Ig binds to CHO.PD-1 cells (solid circle) but not untransfectedCHO cells (gray triangle).

FIG. 2 shows that B7-DC-Ig competes with B7-H1 for binding to PD-1.Unlabeled B7-DC-Ig at various concentrations was first incubated with aCHO cell line constitutively expressing PD-1 before adding labeledB7-H1-Ig to the cell mixture. The median fluorescence intensity (MFI) ofB7-H1-Ig (y-axis) is shown as a function of the concentration ofunlabeled B7-DC-Ig competitor (x-axis) added. As the concentration ofunlabeled B7-DC-Ig is increased the amount of labeled B7-H1-Ig bound toCHO cells decreases, demonstrating that B7-DC-Ig competes with B7-H1 forbinding to PD-1.

FIG. 3 shows the results of experiments wherein the combination ofcyclophosphamide (CTX or Cytoxan®) and dimeric murine B7-DC-Ig resultedin eradication of established CT26 tumors (colon carcinoma) in mice.Graph A shows tumor volume (mm³) versus days post tumor challenge inmice treated with 100 mg/kg of CTX on Day 10 while Graph B shows tumorvolume (mm³) versus days post tumor challenge in mice treated with CTXon Day 10 followed a day later by the first B7-DC-Ig administration.Each line in each graph represents one mouse. Black arrow stands forB7-DC-Ig administration. Graph C shows average tumor volume.

FIG. 4 shows the results of experiments wherein the combination of CTXand dimeric murine B7-DC-Ig eradicated established CT26 tumors (coloncarcinoma) in mice and protected against re-challenge with CT26. Micethat were treated with CTX and B7-DC-Ig and found to be free of tumorgrowth on day 44 following tumor inoculation were rechallenged withtumors. The mice were later rechallenged again on on Day 70. None of themice displayed tumor growth by day 100.

FIG. 5 shows CTX and B7-DC-Ig treatment resulted in generation of tumorspecific memory CTL. Mice eradicated established CT26 subcutenous tumorspost CTX and B7-DC-Ig treatment were re-challenged with CT26 cells.Seven days later, splenocytes were isolated and pulsed with eitherovalbumin, an irrelevant peptide, or AH1, a CT26 specific peptide. Cellswere stained with anti-CD8 antibody first followed by intracellularstaining with anti-IFNγ antibody prior to FACS analysis.

FIG. 6 shows the effects of different doses of B7-DC-Ig in combinationwith CTX on the eradication of established CT26 tumors in mice. Balb/Cmice at age of 9 to 11 weeks were implanted subcutaneously with 1E05CT26 cells. On Day 9, mice were injected IP with 100 mg/kg of CTX.Twenty four hours later, on Day 10, mice were treated with 30, 100, or300 ug of B7-DC-Ig followed by 2 injections every week up to total 8treatments. Tumor growth was measured two times per week.

FIG. 7 shows the results of experiments wherein the combination of CTXand anti-PD-1 antibody resulted in eradication of established CT26tumors (colon carcinoma) in mice. Graph A shows tumor volume (mm³)versus days post tumor challenge in untreated mice (i.e., mice treatedwith vehicle alone), Graph B shows tumor volume (mm³) versus days posttumor challenge in mice treated with anti-PD-1 alone starting on Day 11at 300 μg per injection, 3 times per week, up to 12 injections and GraphC shows tumor volume (mm³) versus days post tumor challenge in micetreated with CTX on Day 11 and the first anti-PD-1 administration on Day12 at 300 μg per injection, 3 times per week, up to 12 injections. Eachline in each graph represents one mouse. Black arrow stands foranti-PD-1 administration.

FIG. 8 shows the results of experiments wherein the combination of CTXand anti-CTLA4 antibody resulted in eradication of established CT26tumors (colon carcinoma) in mice. Here, Graph A shows tumor volume (mm³)versus days post tumor challenge in mice treated with 100 mg/kg of CTXon Day 11 while Graph B shows tumor volume (mm³) versus days post tumorchallenge in mice treated with CTX on Day 11 and anti-CTLA4 on Day 12 at100 μg per injection, 2 times per week, up to 8 injections. Each line ineach graph represents one mouse. Black arrow stands for anti-CTLA-4administration.

FIG. 9 shows the results of experiments wherein Balb/C mice at age of 9to 11 weeks of age were implanted with 1×10⁵ CT26 cells subcutaneously.On Day 9, mice were injected with 100 mg/kg of CTX, IP. Twenty fourhours later, on Day 10, mice were treated with 100 ug of B7-DC-Ig. Therewere 5 groups: naïve mice that did not receive any tumor cells, vehicleinjected, CTX alone, CTX+B7-DC-Ig or B7-DC-Ig alone. Two naïve mice and4 mice from other groups were removed from the study on Day 11 (2 dayspost CTX) and Day 16 (7 days post CTX) for T cell analysis. Left panelshows on Day 11, 2 days post CTX injection, Treg in the spleen of themice with CTX treatment was significantly lower than the one in the micewith tumor implantation and injected with vehicle. Right panel showsthat on Day 16, 7 days post CTX and 6 days post B7-DC-Ig treatment,B7-DC-Ig significantly lowered the CD4+ T cells expressing high PD-1.This was observed in both the B7-DC-Ig treated and CTX+B7-DC-Ig treatedmice. Mice implanted with tumor cells intended to have more PD-1+/CD4+ Tcells in the draining LN compared with naïve mice.

FIG. 10 shows the results of experiments wherein the combination of CTXand B7-DC-Ig resulted in increased survival in mice with tail veininjection of a mouse prostate tumor cell line. SP-1 cells were isolatedfrom mouse lungs that were metastasized from TRAMP prostate tumor cellinjection. B10.D2 mice were first injected with 3×10⁵ SP-1 cells viatail vein injection. On Day 5, 12 and 19, mice were injected with 50mg/kg of CTX where was indicated. On Day 6, 13 and 20, mice wereadministered with 5 mg/kg of B7-DC-Ig were it was indicated. Here, “NT”refers to “not treated”.

FIG. 11. Balb/C mice at age of 11-13 weeks were given isolated hepaticmetastases using a hemispleen injection technique. The spleens ofanesthetized mice were divided into two halves and the halves wereclipped. CT26 cells (1E05) were injected into one hemispleen, and after30 seconds, that hemispleen was resected and the splenic draining veinwas clipped. On Day 10, mice received 1 injection of CTX at 50 mg/kg,IP. Twenty four hours later, on Day 11, mice were treated withrecombinant Listeria carrying AH1 peptide, an immunodominant epitope ofCT26, at 0.1×LD₅₀ (1×10⁷ CFU), then on Day 14 and 17. Mice were alsotreated with B7-DC-Ig on Day 11 and then on Day 18. Mouse overallsurvival was monitored.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. In particular, the followingterms and phrases have the following meaning.

The term “inhibitory signal transduction” is intended to mean any signaltransduction having the effect of abolishing, or otherwise reducing, Tcell responses against an antigen, whether by reducing T cellproliferation or by any other inhibitory mechanism, whereby the extentor duration of an immunogenic T cell response is decreased. Suchinhibitory signal transduction may be due to PD-1 binding to a naturalligand, such as binding of PD-1 by B7-H1 or some other member of thisclass of ligands, B7-DC, or may be due to binding of CTLA4 to ligands,such as B7-1 or B7-2. In general, compounds of the invention reduce suchinhibitory signal transduction and include, but are not limited to, PD-1antagonists and CTLA4 antagonists.

The term “PD-1 antagonist” means any molecule that attenuates inhibitorysignal transduction mediated by PD-1, found on the surface of T cells, Bcells, natural killer (NK) cells, monocytes, DC, and macrophages. Suchan antagonist includes a molecule that disrupts any inhibitory signalgenerated by a PD-1 molecule on a T cell. In specific examples of theinvention, a PD-1 antagonist is a molecule that inhibits, reduces,abolishes or otherwise reduces inhibitory signal transduction throughthe PD-1 receptor signaling pathway. Such decrease may result where: (i)the PD-1 antagonist of the invention binds to a PD-1 receptor withouttriggering signal transduction, to reduce or block inhibitory signaltransduction; (ii) the PD-1 antagonist binds to a ligand (e.g. anagonist) of the PD-1 receptor, preventing its binding thereto (forexample, where said agonist is B7-H1); (iii) the PD-1 antagonist bindsto, or otherwise inhibits the activity of, a molecule that is part of aregulatory chain that, when not inhibited, has the result of stimulatingor otherwise facilitating PD-1 inhibitory signal transduction; or (iv)the PD-1 antagonist inhibits expression of a PD-1 receptor or expressionligand thereof, especially by reducing or abolishing expression of oneor more genes encoding PD-1 or one or more of its natural ligands. Thus,a PD-1 antagonist of the invention is a molecule that effects a decreasein PD-1 inhibitory signal transduction, thereby increasing T cellresponse to one or more antigens.

As used herein, the term “CTLA4 antagonist” means a compound thatreduces CTLA4-mediated inhibition of T cell reactions. For example, inan T cell, CTLA4 delivers an inhibitory impulse upon binding of B7ligands, such B7-1 and B7-2. A CTLA4 antagonist is one that disruptsbinding of said ligands to CTLA4 on activated T cells. In oneembodiment, the antagonist is an anti-CTLA4 antibody that binds CTLA4 toprevent ligand binding. I

As used herein, the term “active fragment” refers to a portion of anatural polypeptide, or a polypeptide with high sequence homology (forexample, at least 80%, 85%, 90%, 95%, 98%, or 99% amino acid sequenceidentity) to a natural polypeptide and that exhibits PD-1 antagonistactivity, for example, by binding PD-1 or by binding to a ligand ofPD-1. In preferred embodiments, such a fragment would consist of theextracellular domain (ECD) of a B7-DC protein that binds to PD-1, suchas SEQ ID NO: 3, preferably amino acids 20 to 221 thereof. In the caseof PD-1 polypeptide, an active fragment would be a portion of saidpolypeptide comprising a binding domain that binds to a natural ligandof PD-1 to prevent stimulation of PD-1 mediated inhibitory signaltransduction by said ligand. Active fragments may be identified by theirability to compete with the molecule they are derived from for bindingto a natural binding site. For example, active fragments will competewith wild-type B7-DC for binding to PD-1.

With respect to an antibody, the term “active fragment” means an antigenbinding portion of an antibody that is less than an entireimmunoglobulin. Such fragments include Fab and F(ab₂)′ fragments,capable of reacting with and binding to any of the polypeptidesdisclosed herein as being receptors or ligands. These Fab and F(ab′)₂fragments lack the Fc portion of an intact antibody, clear more rapidlyfrom the circulation, and may have less non-specific tissue binding thanan intact antibody (Wahl et al., J. Nuc. Med. 24:316-325 (1983)). Alsoincluded are Fv fragments (Hochman, J. et al. (1973) Biochemistry12:1130-1135; Sharon, J. et al. (1976) Biochemistry 15:1591-1594). Thesevarious fragments are produced using conventional techniques such asprotease cleavage or chemical cleavage (see, e.g., Rousseaux et al.,Meth. Enzymol., 121:663-69 (1986)).

As used herein, the term “soluble portion” of a PD-1 antagonist meansthat portion of the full length polypeptide that does not include anypart of the transmembrane portion or segment. For example, with respectto B7-DC, a soluble portion would include the extracellular portion(with or without the N-terminal signal sequence) but would not includeany part of the transmembrane portion (or, at least, not enough toreduce solubility). Thus, the ECD of human B7-DC is shown as SEQ ID NO:3 and consists of both the IgV-like and IgC-like domains of the fulllength molecule (i.e., amino acids 20-221 of the full length sequence(SEQ ID NO: 1).

As used herein, a “co-stimulatory polypeptide” is a polypeptide that,upon interaction with a cell-surface molecule on T cells, modulates theactivity of the T cell. Thus, the response of the T cell can be aneffector (e.g., CTL or antibody-producing B cell) response, a helperresponse providing help for one or more effector (e.g., CTL orantibody-producing B cell) responses, or a suppressive response.

As used herein, the term “treatment regimen” refers to a treatment of adisease or a method for achieving a desired physiological change, suchas increased or decreased response of the immune system to an antigen orimmunogen, such as an increase or decrease in the number or activity ofone or more cells, or cell types, that are involved in such response,wherein said treatment or method comprises administering to an animal,such as a mammal, especially a human being, a sufficient amount of twoor more chemical agents or components of said regimen to effectivelytreat a disease or to produce said physiological change, wherein saidchemical agents or components are administered together, such as part ofthe same composition, or administered separately and independently atthe same time or at different times (i.e., administration of each agentor component is separated by a finite period of time from one or more ofthe agents or components) and where administration of said one or moreagents or components achieves a result greater than that of any of saidagents or components when administered alone or in isolation.

As used herein the term “isolated” is meant to describe a compound ofinterest (e.g., either a polynucleotide or a polypeptide) that is in anenvironment different from that in which the compound naturally occurse.g. separated from its natural milieu such as by concentrating apeptide to a concentration at which it is not found in nature.“Isolated” is meant to include compounds that are within samples thatare substantially enriched for the compound of interest and/or in whichthe compound of interest is partially or substantially purified.

As used herein, the term “polypeptide” refers to a chain of amino acidsof any length, regardless of modification (e.g., phosphorylation orglycosylation). A polypeptide of the present invention may be arecombinant polypeptide, a natural polypeptide or a syntheticpolypeptide, preferably a recombinant polypeptide.

As used herein, a “variant” polypeptide contains at least one amino acidsequence alteration as compared to the amino acid sequence of thecorresponding wild-type polypeptide.

As used herein, an “amino acid sequence alteration” can be, for example,a substitution, a deletion, or an insertion of one or more amino acids.

As used herein, the terms “portion,” “segment,” and “fragment,” whenused in relation to polypeptides, refer to a continuous sequence ofresidues, such as amino acid residues, which sequence forms a subset ofa larger sequence. For example, if a polypeptide were subjected totreatment with any of the common endopeptidases, such as trypsin orchymotrypsin, the oligopeptides resulting from such treatment wouldrepresent portions, segments or fragments of the starting polypeptide. A“fragment” of a polypeptide thus refers to any subset of the polypeptidethat is a shorter polypeptide of the full length protein. Generally,fragments will be five or more amino acids in length.

A derivative, analog or homolog, of a polypeptide (or fragment thereof)of the invention may be (i) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code, or(ii) one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchderivatives and analogs are deemed to be within the scope of thoseskilled in the art from the teachings herein.

As used herein, “valency” refers to the number of binding sitesavailable per molecule.

In accordance with the present invention, the term “percent identity” or“percent identical,” when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The Percent Identity isthen determined according to the following formula;Percent Identity=100[1−(C/R)]wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence wherein (I) each base oramino acid in the Reference Sequence that does not have a correspondingaligned base or amino acid in the Compared Sequence and (ii) each gap inthe Reference Sequence and (iii) each aligned base or amino acid in theReference Sequence that is different from an aligned base or amino acidin the Compared Sequence, constitutes a difference; and R is the numberof bases or amino acids in the Reference Sequence over the length of thealignment with the Compared Sequence with any gap created in theReference Sequence also being counted as a base or amino acid. If analignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which thehereinabove calculated Percent Identity is less than the specifiedPercent Identity.

As used herein, the term “conservative amino acid substitution” means asubstitution wherein the substituted amino acid has similar structuralor chemical properties, and “non-conservative” amino acid substitutionsare those in which the charge, hydrophobicity, or bulk of thesubstituted amino acid is significantly altered. Non-conservativesubstitutions will differ more significantly in their effect onmaintaining (a) the structure of the peptide backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thebulk of the side chain. Examples of conservative amino acidsubstitutions include those in which the substitution is within one ofthe five following groups: 1) small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); 2) polar, negatively chargedresidues and their amides (Asp, Asn, Glu, Gln); polar, positivelycharged residues (His, Arg, Lys); large aliphatic, nonpolar residues(Met, Leu, Ile, Val, Cys); and large aromatic resides (Phe, Tyr, Trp).Examples of non-conservative amino acid substitutions are those where 1)a hydrophilic residue, e.g., seryl or threonyl, is substituted for (orby) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl,or alanyl; 2) a cysteine or proline is substituted for (or by) any otherresidue; 3) a residue having an electropositive side chain, e.g., lysyl,arginyl, or histidyl, is substituted for (or by) an electronegativeresidue, e.g., glutamyl or aspartyl; or 4) a residue having a bulky sidechain, e.g., phenylalanine, is substituted for (or by) a residue thatdoes not have a side chain, e.g., glycine.

The terms “individual”, “host”, “subject”, and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, primates, for example, human beings, as well as rodents,such as mice and rats, and other laboratory animals.

As used herein the term “effective amount” or “therapeutically effectiveamount” means a dosage sufficient to treat, inhibit, or alleviate one ormore symptoms of a disease state being treated or to otherwise provide adesired pharmacologic and/or physiologic effect, especially enhancing Tcell response to a selected antigen. The precise dosage will varyaccording to a variety of factors such as subject-dependent variables(e.g., age, immune system health, etc.), the disease, and the treatmentbeing administered.

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the therapeuticcompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

The term “antibody” is meant to include both intact molecules as well asfragments thereof that include the antigen-binding site. Whole antibodystructure is often given as H₂L₂ and refers to the fact that antibodiescommonly comprise 2 light (L) amino acid chains and 2 heavy (H) aminoacid chains. Both chains have regions capable of interacting with astructurally complementary antigenic target. The regions interactingwith the target are referred to as “variable” or “V” regions and arecharacterized by differences in amino acid sequence from antibodies ofdifferent antigenic specificity. The variable regions of either H or Lchains contains the amino acid sequences capable of specifically bindingto antigenic targets. Within these sequences are smaller sequencesdubbed “hypervariable” because of their extreme variability betweenantibodies of differing specificity. Such hypervariable regions are alsoreferred to as “complementarity determining regions” or “CDR” regions.These CDR regions account for the basic specificity of the antibody fora particular antigenic determinant structure. The CDRs representnon-contiguous stretches of amino acids within the variable regions but,regardless of species, the positional locations of these critical aminoacid sequences within the variable heavy and light chain regions havebeen found to have similar locations within the amino acid sequences ofthe variable chains. The variable heavy and light chains of allantibodies each have 3 CDR regions, each non-contiguous with the others(termed L1, L2, L3, H1, H2, H3) for the respective light (L) and heavy(H) chains. The accepted CDR regions have been described by Kabat et al,J. Biol. Chem. 252:6609-6616 (1977). The antibodies disclosed accordingto the invention may also be wholly synthetic, wherein the polypeptidechains of the antibodies are synthesized and, possibly, optimized forbinding to the polypeptides disclosed herein as being receptors. Suchantibodies may be chimeric or humanized antibodies and may be fullytetrameric in structure, or may be dimeric and comprise only a singleheavy and a single light chain.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a treatment regimen, or combinationtherapy, for treating disease in mammals comprising a compound thatreduces or abolishes inhibitory signal transduction in T cells,preferably human T cells, administered in conjunction with apotentiating agent to increase an immune response.

The methods of the invention also relate to the use of broad spectrumimmunomodulators and compositions of these. In general, the increased Tcell response resulting from these methods is greater than any increasedT cell response resulting from administering the same dose of either ofsaid PD-1 antagonist or said potentiating agent alone.

The disclosed compositions and regimens are useful to stimulate orenhance immune responses involving T cells. Thus, the methods of theinvention are most useful in treating a disease condition that wouldbenefit from an increase in T cell activity and where the increased Tcell response is necessary or sufficient to treat said disease, eventhough the disease is not specifically caused or aggravated by a reducedT cell response. In a preferred embodiment, the type of disease to betreated or prevented is a malignant tumor or a chronic infectiousdisease caused by a bacterium, virus, protozoan, helminth, or otherintracellular microbial pathogen that is attacked, i.e., by cytotoxic Tlymphocytes. Activation of T cells using the disclosed compositions isalso advantageous to treat or prevent conditions characterized byimmunosuppression.

In accordance with the present invention, the T cell response can beregulated by molecules that bind to receptors on the T cell surface andmolecules that bind to ligands of such receptors. In the case of PD-1,molecules that bind PD-1 to reduce its inhibitory effect and/ormolecules that bind one or more PD-1 ligands to reduce their ability tobind PD-1 have the effect of reducing the ability of PD-1 to inhibit Tcell response, thereby increasing this response and the immunologicaleffects thereof.

A. PD-1 Receptor Antagonists

Compositions containing antagonists of PD-1 receptors are provided andinclude compounds or agents that either bind to and block a ligand ofPD-1 to interfere with or inhibit the binding of the ligand to the PD-1receptor, or bind directly to and block the PD-1 receptor withoutinducing inhibitory signal transduction through the PD-1 receptor. Inanother embodiment, the PD-1 receptor antagonist binds directly to thePD-1 receptor without triggering inhibitory signal transduction and alsobinds to a ligand of the PD-1 receptor to reduce or inhibit the ligandfrom triggering signal transduction through the PD-1 receptor. Byreducing the number and/or amount of ligands that bind to PD-1 receptorand trigger the transduction of an inhibitory signal, fewer cells areattenuated by the negative signal delivered by PD-1 signal transductionand a more robust immune response can be achieved.

In accordance with the present invention, PD-1 signaling requiresbinding to a PD-1 ligand (such as B7-H1 or B7-DC) in close proximity toa peptide antigen presented by major histocompatibility complex (MHC)(see, for example, Freeman Proc. Natl. Acad. Sci. U.S.A 105:10275-10276(2008)). Therefore, proteins, antibodies or small molecules that preventco-ligation of PD-1 and TCR on the T cell membrane are useful PD-1antagonists contemplated by this invention.

Exemplary PD-1 receptor antagonists include, but are not limited toB7-DC polypeptides, including homologs and variants of these, as well asactive fragments of any of the foregoing, and fusion proteins thatincorporate any of these. In a preferred embodiment, the fusion proteincomprises the soluble portion of B7-DC coupled to the Fc portion of anantibody, such as human IgG, and does not incorporate all or part of thetransmembrane portion of human B7-DC. The PD-1 receptor antagonists canalso be small molecule antagonists or antibodies that reduce orinterfere with PD-1 receptor signal transduction by binding to ligandsof PD-1 or to PD-1 itself, especially where co-ligation of PD-1 with TCRdoes not follow such binding, thereby not triggering inhibitory signaltransduction through the PD-1 receptor.

The PD-1 receptor antagonists provided herein are generally useful invivo and ex vivo as immune response-stimulating therapeutics. Ingeneral, the disclosed antagonist compositions are useful for treating asubject having or being predisposed to any disease or disorder to whichthe subject's immune system mounts an immune response.

1. B7-DC Polypeptides

In certain embodiments, B7-DC proteins can be used as PD-1 receptorantagonists. B7-DC is a natural ligand of PD-1 and binds to PD-1 withhigher affinity than B7-H1, and can thus inhibit B7-H1:PD-1interactions. Suitable B7-DC polypeptides, including variants, homologsand fragments thereof, can be obtained from the following full lengthhuman B7-DC polypeptides with (SEQ ID NO:1) or without (SEQ ID NO:2) theendogenous signal peptide.

(SEQ ID NO: 1)MIFLLLMLSL ELQLHQIAAL FTVTVPKELY IIEHGSNVTL ECNFDTGSHV NLGAITASLQ  60KVENDTSPHR ERATLLEEQL PLGKASFHIP QVQVRDEGQY QCIIIYGVAW DYKYLTLKVK 120ASYRKINTHI LKVPETDEVE LTCQATGYPL AEVSWPNVSV PANTSHSRTP EGLYQVTSVL 180RLKPPPGRNF SCVFWNTHVR ELTLASIDLQ SQMEPRTHPT WLLHIFIPFC IIAFIFIATV 240IALRKQLCQK LYSSKDTTKR PVTTTKREVN SAI 273 (SEQ ID NO: 2)LFTVTVPKEL YIIEHGSNVT LECNFDTGSH VNLGAITASL QKVENDTSPH RERATLLEEQ  60LPLGKASFHI PQVQVRDEGQ YQCIIIYGVA WDYKYLTLKV KASYRKINTH ILKVPETDEV 120ELTCQATGYP LAEVSWPNVS VPANTSHSRT PEGLYQVTSV LRLKPPPGRN FSCVFWNTHV 180RELTLASIDL QSQMEPRTHP TWLLHIFIPF CITAFIFIAT VIALRKQLCQ KLYSSKDTTK 240RPVTTTKREV NSAI 254

The B7 family of molecules, including B7-DC, are expressed at the cellsurface with a membrane proximal constant IgC domain and a membranedistal IgV domain. Receptors for these ligands share a commonextracellular IgV-like domain. Interactions of receptor-ligand pairs aremediated predominantly through residues in the IgV domains of theligands and receptors. In general, IgV domains are described as havingtwo sheets that each contains a layer of β-strands. These β-strands arereferred to as A′, B, C, C′, C″, D, E, F and G. The structure of suchpolypeptides has been described in the literature (See Molnar et al.,Crystal structure of the complex between programmed death-1 (PD-1) andits ligand PD-L2, PNAS, Vol. 105, pp. 10483-10488 (29 Jul. 2008)).

B7-DC, a transmembrane protein, in its monomeric form, comprises IgV andIgC domains that make up the extracellular portion of the molecule (theextracellular domain, or ECD), with the IgV-like domain beingresponsible, in whole or in part, for PD-1 binding as well as otherfunctions recited in the methods of the invention. For the humanprotein, the IgV domain is characterized in that it possesses adisulfide bond linking the B and F strands (referred to above), whichappears to be characteristic of many IgV domains and possesses a similarthree-dimensional structure with the IgV domains of both B7-1 and B7-2(see Molnar et al. (2008), supra).

In one embodiment the B7-DC variant polypeptides contain amino acidalterations (i.e., substitutions, deletions or insertions) within one ormore of these β-strands in any possible combination. In anotherembodiment, B7-DC variants contain one or more amino acid alterations(i.e., substitutions, deletions or insertions) within the A′, C, C′, C″,D, E, F or G β-strands. In a preferred embodiment B7-DC variants containone or more amino acid alterations in the G β-strand. In anotherembodiment, variant B7-DC polypeptide fragments include the IgC and IgVdomains of B7-DC. In another embodiment, variant B7-DC polypeptidefragments include the IgV domain of B7-DC.

Human and mouse B7-DC proteins contain a short intracytoplasmic domain,a single transmembrane domain and an extracellular domain. Theextracellular domain contains two Ig domains; a membrane proximal IgCdomain and a membrane distal IgV domain. Useful fragments of variantB7-DC polypeptides include soluble fragments. Soluble B7-DC fragmentsare fragments of B7-DC that may be shed, secreted or otherwise extractedfrom the producing cells. In one embodiment, variant B7-DC polypeptidefragments include the entire extracellular domain of B7-DC. Theextracellular domain of B7-DC includes amino acids from about 20 toabout amino acid 221 of murine or human B7-DC or active fragmentsthereof. In another embodiment, variant B7-DC polypeptide fragmentsinclude the IgC and IgV domains of B7-DC. In another embodiment, variantB7-DC polypeptide fragments include the IgV domain of B7-DC.

PD-1 signaling is thought to require binding to a PD-1 ligand (typicallyB7-H1) in close proximity to a peptide antigen presented by majorhistocompatibility complex (MHC) (Freeman Proc. Natl. Acad. Sci. U.S. A105:10275-10276 (2008)). Therefore, proteins, antibodies or smallmolecules that prevent co-ligation of PD-1 and TCR on the T cellmembrane are useful PD-1 antagonists contemplated by this invention.

The PD-1 antagonist useful in the methods and compositions of theinvention include fragments of the B7-DC protein incorporating the ECD.Alternatively, the fragments of B7-DC include part of the extracellulardomain that comprise the an IgV or IgV-like domain, preferably aminoacids 20-221, more preferably 20-121, that are sufficient to bind to thePD-1 receptor to interfere with, or prevent, or otherwise reduceinhibitory signal transduction through the PD-1 receptor. In a preferredembodiment the B7-DC fragment competes with B7-H1 for binding to PD-1receptors.

In one embodiment, variant B7-DC polypeptide fragments may contain aregion of the polypeptide that is important for binding to PD-1. Thesepolypeptide fragments may be useful to compete for binding to PD-1 andto prevent native B7-DC from binding to PD-1. By competing for bindingto PD-1, these fragments may be useful to enhance an immune response, asinhibiting interactions of B7-H1 and B7-DC with PD-1 inhibits thesuppression of immune responses that would otherwise occur. Apolypeptide fragment of mouse or human B7-DC that could competitivelybind to PD-1 can contain, for example, amino acids 101-108 or 110-114.The binding of wild-type B7-DC to PD-1 typically is inhibited by atleast 50 percent, 60 percent, 70 percent, 75 percent, 80 percent, 90percent, 95 percent, or more than 95 percent as compared to the level ofbinding of wild-type B7-DC to PD-1 in the absence of a fragment of saidwild-type B7-DC. Exemplary B7-DC fragments useful in the methods and/orcompositions of the invention include, but are in no way limited to, thefollowing B7-DC extracellular domains:

Human B7-DC extracellular domain (ECD): (SEQ ID NO: 3)LFTVTVPKEL YIIEHGSNVT LECNFDTGSH VNLGAITASL QKVENDTSPH RERATLLEEQ  60LPLGKASFHI PQVQVRDEGQ YQCIIIYGVA WDYKYLTLKV KASYRKINTH ILKVPETDEV 120ELTCQATGYP LAEVSWPNVS VPANTSHSRT PEGLYQVTSV LRLKPPPGRN FSCVFWNTHV 180RELTLASIDL QSQMEPRTHP TW 202 and murine B7-DC ECD: (SEQ ID NO: 4)LFTVTAPKEV YTVDVGSSVS LECDFDRREC TELEGIRASL QKVENDTSLQ SERATLLEEQ  60LPLGKALFHI PSVQVRDSGQ YRCLVICGAA WDYKYLTVKV KASYMRIDTR ILEVPGTGEV 120QLTCQARGYP LAEVSWQNVS VPANTSHIRT PEGLYQVTSV LRLKPQPSRN FSCMFWNAHM 180KELTSAIIDP LSRMEPKVPR TW 202 Cynomolgus monkey B7-DC ECD:(Seq ID NO: 15)LFTVTVPKEL YIIEHGSNVT LECNFDTGSH VNLGAITASL QKVENDTSPH RERATLLEEQ  60LPLGKASFHI PQVQVRDEGQ YQCIIIYGVA WDYKYLTLKV KASYRKINTH ILKVPETDEV 120ELTCQATGYP LAEVSWPNVS VPANTSHSRT PEGLYQVTSV LRLKPPPGRN FSCVFWNTHV 180RFLTLASTDL QSQMEPRTHP TW 202

Numerous other primate sequences useful in the methods and compositionsof the invention are provided in Onlamoon et al., Immunology, Vol. 124,pp. 277-293 (2008).

A PD-1 antagonist useful in the compositions and methods of theinvention also includes a fusion protein (as described below) thatcomprises first and second polypeptide portions, wherein said fusionprotein, or at least the first polypeptide portion thereof, possessesPD-1 antagonist activity, especially where said fusion protein binds toand blocks PD-1 or binds to and blocks a ligand of PD-1. The firstpolypeptide portion of such fusion protein can comprise, or consist of,any of the PD-1 antagonistic polypeptides, or PD-1 binding fragmentsthereof, otherwise recited herein for use as PD-1 antagonists in themethods of the invention. In a preferred embodiment of such a fusionprotein, the recited first polypeptide portion is N-terminal to therecited second polypeptide portion. In a separate embodiment, therecited first polypeptide portion is linked to the recited secondpolypeptide portion by an oligopeptide in addition to the amino acidscomposing the recited first and second polypeptide portions, where saidlinking amino acids do not substantially decrease the PD-1 antagonistactivity of said fusion protein.

In a preferred dimeric fusion protein, the dimer results from thecovalent bonding of Cys residues in the CH regions of two of the Igheavy chains that are the same Cys residues that are disulfide linked indimerized normal Ig heavy chains.

A large number of polypeptide sequences that are routinely used asfusion protein binding partners are well known in the art. Examples ofuseful polypeptide binding partners include, but are not limited to,green fluorescent protein (GFP), glutathione S-transferase (GST),polyhistidine, myc, hemaglutinin, Flag™ tag (Kodak, New Haven, Conn.),maltose E binding protein and protein A.

Still another embodiment provides a tetramer construct having a BirAsubstrate fused to the extracellular domain of a variant B7-DCpolypeptide. Methods for making tetramer constructs are known in the art(see Pertovas, et al., J. Exp. Med., 203:2281 (2006)).

Exemplary murine B7-DC fusion proteins contain amino acids 20-221 ofmurine B7-DC fused to amino acids 237-469 of murine IgG2a (CAA49868). Inone non-limiting example, human B7-DC fusion proteins contain aminoacids 20-221 of human B7-DC fused to amino acids 245-476 of human IgG1(AAA02914). The signal peptides for B7-DC fusion proteins include theendogenous signal peptides or any other signal peptide that facilitatessecretion of the fusion protein from a host. In another embodiment, thefirst polypeptide would include only the IgV domain. Other embodimentsmay comprise the hinge and Fc domain of an IgG antibody, such IgG1, withnone of the variable region present. Other embodiments include use ofthe hinge and Fc region of IgG2 or IgG4, especially having an N297Q orother mutation that reduces effector function.

In accordance with the methods and compositions of the invention, thepolypeptide useful as a PD-1 antagonist, or the first polypeptideportion of a fusion protein useful as a PD-1 antagonist, comprises anamino acid sequence that has at least 60%, or at least 65%, or at least70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%,or at least 95%, or at least 99%, identity to amino acids 1-221 of SEQID NO: 1, preferably amino acids 20-221 of SEQ ID NO: 1, or amino acids26-221 of SEQ ID NO: 1, or amino acids 1-202 of SEQ ID NO: 3 or 4, morepreferably amino acids 20-121 of SEQ ID NO: 1 or amino acids 1-102 ofSEQ ID NO: 3 or 4.

In one embodiment, a polypeptide useful as a PD-1 antagonist, or thefirst polypeptide portion of a fusion protein useful as a PD-1antagonist, consists of amino acids 1-221 of SEQ ID NO: 1, or consistsof amino acids 20-221 of SEQ ID NO: 1, or consists of amino acids 26-221of SEQ ID NO: 1, or consists of amino acids 1-202 of SEQ ID NO: 3 or 4.In one embodiment (SEQ ID NO: 2), it does not comprise amino acids 1-19of SEQ ID NO: 1.

In other specific examples, a PD-1 antagonist polypeptide, or firstpolypeptide portion of a PD-1 antagonist fusion protein, comprises theamino acid sequence 20-121 of SEQ ID NO: 1, preferably where itcomprises the amino acid sequence WDYKY at residues 110-114 thereof, orwhere it comprises amino acids 1-102 of SEQ ID NO: 3, preferably whereit comprises the amino acid sequence WDYKY at residues 91-95 thereof.

In a preferred embodiment, such percent identities are achieved byreliance on conservative amino acid substitutions as defined elsewhereherein.

In one such embodiment, the PD-1 antagonist polypeptide, or firstpolypeptide portion of a PD-1 antagonist fusion protein, does notcomprise amino acids 1-19 of SEQ ID NO: 1, or does not comprise anyportion of a transmembrane domain, especially not the entire suchdomain, or does not comprise any portion of the intracellular (orsoluble) domain, especially not the entire such domain, of a PD-1 ligandor other PD-1 antagonist protein. In a preferred embodiment, suchantagonist, or first polypeptide portion, comprises only theextracellular domain (ECD) of SEQ ID NO:1 and is thus comprised only ofa soluble portion of the polypeptide of said sequence, or a fragment ofsaid soluble portion.

In other such embodiments, the PD-1 antagonist polypeptide, or firstpolypeptide portion of a PD-1 antagonist fusion protein, comprises theIgV domain, or IgV-like domain, or PD-1 binding fragment thereof, of aPD-1 ligand, or consists of the IgV domain, or IgV-like domain, or PD-1binding fragment thereof, of a PD-1 ligand. In specific examples, suchPD-1 ligand is a wild-type B7-DC or B7-H1 molecule, preferably mouse orprimate, preferably human, wild-type B7-DC or B7-H1 molecule.

In other such embodiments, the PD-1 antagonist polypeptide, or firstpolypeptide portion of a PD-1 antagonist fusion protein, a PD-1 bindingfragment of the IgV domain, or IgV-like domain, of a PD-1 ligand,especially where IgV domain, or IgV-like domain, consists of amino acids20-121 of SEQ ID NO: 1 or amino acids 1-102 of SEQ ID NO: 3.

A PD-1 antagonist of the invention also includes a PD-1 binding fragmentof amino acids 20-121 of SEQ ID NO: 1 (human full length), or aminoacids 1-102 of SEQ ID NO: 3 (extracellular domain or ECD).

In specific embodiments thereof, the polypeptide or PD-1 bindingfragment also incorporates amino acids WDYKY at residues 110-114 of SEQID NO: 1 or WDYKY at residues 91-95 of SEQ ID NO: 3. By way ofnon-limiting examples, such a PD-1 binding fragment comprises at least10, or at least 20, or at least 30, or at least 40, or at least 50, orat least 60, or at least 70, or at least 75, or at least 80, or at least85, or at least 90, or at least 95, or at least 100 contiguous aminoacids of the sequence of amino acids 20-121 of SEQ ID NO: 1, wherein apreferred embodiment of each such PD-1 binding fragment would compriseas a sub-fragment the amino acids WDYKY found at residues 110-114 of SEQID NO: 1 or WDYKY at residues 91-95 of SEQ ID NO: 3.

Other preferred polypeptides and PD-1 binding fragments specificallycontemplated by the invention include the polypeptide sequence of aminoacids 20-121 of SEQ ID NO: 1 (human full length) and PD-1 bindingfragments thereof, wherein, in such polypeptide or PD-1 bindingfragment, a cysteine is present at residues 42 and/or 102, with acysteine at both positions being preferred, and/or wherein aphenylalanine is present at residue 21, and/or wherein a glutamic acidis present at residue 28, and/or wherein a threonine, and/or wherein aglutamine is present at residue 60, and/or wherein a glutamic acid ispresent at residue 101, and/or wherein isoleucine is present at residue103, and/or wherein an isoleucine is present at residue 105, and/orwherein a glycine is present at residue 107, and/or wherein valine ispresent at residue 108, and/or wherein a tryptophan is present atresidue 110, and/or wherein aspartic acid is present at residue 111,and/or wherein a tyrosine is present at residue 112, and/or wherein alysine is present at residue 113, and/or wherein a tyrosine is presentat residue 114, provided that, in the case of PD-1 binding fragments,said fragment is large enough to include such amino acid positions.

Additional preferred polypeptides and PD-1 binding fragmentsspecifically contemplated by the invention include the polypeptidesequence of amino acids 1-102 of SEQ ID NO: 3 (human ECD) or SEQ ID NO:4 (murine ECD) and PD-1 binding fragments thereof, wherein, in suchpolypeptide or PD-1 binding fragment, a cysteine is present at residues23 and/or 83, with a cysteine at both positions being preferred, and/orwherein a phenylalanine is present at residue 2, and/or wherein aglutamic acid is present at residue 9, and/or wherein a threonine orarginine is present at residue 37, with threonine preferred, and/orwherein a glutamine is present at residue 41, and/or wherein arginine ispresent at residue 82, and/or wherein a leucine is present at residue84, and/or wherein an isoleucine is present at residue 86, and/orwherein a glycine is present at residue 88, and/or wherein an alanine ispresent at residue 89, and/or wherein a tryptophan is present at residue91, and/or wherein a aspartic acid is present at residue 92, and/orwherein a tyrosine is present at residue 93, and/or wherein a lysine ispresent at residue 94, and/or wherein a tyrosine is present at residue95, provided that, in the case of PD-1 binding fragments, said fragmentis large enough to include such amino acid positions.

In additional embodiments, any of the above polypeptides may alsoincorporate portions or fragments, for example, from 1 to 10 contiguousamino acids, drawn from the signal, transmembrane or C-terminal domainsof the B7-DC or 67-H1 polypeptide, such as that of mouse or primate,preferably human.

Such polypeptides and/or PD-1 binding fragments can also be present inany of the fusion proteins of the invention, for example, where suchpolypeptide or PD-1 binding fragment represents the “first polypeptide”of such fusion protein.

In specific examples, the molecule, combined with a potentiating agentfor use in a treatment regimen of the invention, comprises a PD-1binding fragment of amino acids 20-221 of SEQ ID NO: 1. In one suchembodiment, the fragment is from amino acids 20-121 of SEQ ID NO: 1,preferably where the fragment contains amino acids 110-114 of SEQ IDNO: 1. In some embodiments, more than one such fragment is present (asdescribed elsewhere herein) and the molecule comprises at least 2, 3, 4,5 or more fragments of a B7-DC protein, especially where the fragment ispart of, or contains part of, amino acids 20-221 of SEQ ID NO: 1. In apreferred embodiment thereof, at least one said fragment is from aminoacids 20-121 of SEQ ID NO: 1, more preferrably wherein at least one saidfragment includes amino acids 110-114 of SEQ ID NO: 1 (i.e., thesequence WDYKY (SEQ ID NO: 14)). In preferred embodiments, the PD-1binding fragment comprises at least 10, or at least 25, or at least 50,or at least 75, or at least 100 contiguous amino acids in length.

The endogenous human signal peptide has the following sequenceMIFLLLMLSL ELQLHQIAA (SEQ ID NO:5) and represents the first 19 aminoacids of SEQ ID NO: 1. In certain embodiments, the polypeptide fragmentsof B7-DC can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous aminoacids of the endogenous or heterologous signal peptide (which can beused to produce a recombinant B7-DC polypeptide by expression in andsecretion from a transformed cell). It will also be appreciated that auseful B7-DC polypeptide can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10contiguous amino acids of the transmembrane domain of B7-DC, and/or 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids of the cytoplasmicdomain, or combinations thereof provided the B7-DC fragment retains theability to antagonize the PD-1 receptor.

The phenotypes of PD-1 −/− mice provide direct evidence for PD-1 being anegative regulator of immune responses in vivo. In the absence of PD-1,mice on the C57BL/6 background slowly develop a lupus-likeglomerulonephritis and progressive arthritis (Nishimura, et al.,Immunity, 11:141-151 (1999)). PD-1 −/− mice on the BALB/c backgroundrapidly develop a fatal autoimmune dilated cardiomyopathy (Nishimura, etal., Science. 291:319-322 (2001)). However, substantial evidenceindicates that B7-DC can function to costimulate activate T cellresponses. In the presence of suboptimal TCR signals, B7-DC causesincreased proliferation and production of cytokines in vitro (Tseng, etal., J. Exp. Med. 193:839-846 (2001)). On the other hand, in vitrostudies indicate a negative regulatory role for B7-DC in T cellresponses. These seemingly contradictory data are best interpreted byexpression of additional receptors for B7-DC on T cells other than PD-1.

Therefore, B7-DC proteins, variants, fragments and fusions thereof, mayhave the advantage of directly enhancing T cell responses by binding toan unknown receptor that activates the T cell, in addition to enhancingT cell responses by preventing the PD-1 mediated inhibitory signaltransduction.

2. B7-H1 Polypeptides

In another embodiment, the compound for use in combination with apotentiating agent in the treatment regimen of the invention, is, orcomprises, a fragment of a mammalian B7-H1, preferably from mouse orprimate, preferably human, wherein said fragment binds to and blocksPD-1 but does not result in inhibitory signal transduction through PD-1and said fragment is at least 10, or at least 20, or at least 30, or atleast 40, or at least 50, or at least 60, or at least 70, or at least80, or at least 90, or at least 100 contiguous amino acids in length. Inother embodiments, the fragment can be of variable length so long as ithas the function of binding to PD-1 but does not produce inhibitorysignal transduction that results in reduced T cell proliferation. SuchB7-H1 fragments also find use as part of the first polypeptide portionof fusion proteins of the invention.

B7-H1 sequences are as follows:

Human B7-H1 Polypeptide (SEQ ID NO. 16):MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME  60DKNIIQFVHG EEDLKVQHSS YRQRAQLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG 120ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT 180TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNERTH 240LVILGAILLC LGVALTFIFR LRKGRMMDVK KCGIQDTNSK KQSDTHLEET 290 Murine B7-H1(SEQ ID NO: 17)MRIFAGIIFT ACCHLLRAFT ITAPKDLYVV EYGSNVTMEC RFPVERELDL LALVVYWEKE  60DEQVIQFVAG EEDLKPQHSN FRGRASLPKD QLLKGNAALQ ITDVKLQDAG VYCCIISYGG 120ADYKRITLKV NAPYRKINQR ISVDPATSEH ELICQAEGYP EAEVIWTNSD HQPVSGKRSV 280TTSRTEGMLL NVTSSLRVNA TANDVFYCTF WRSQPGQNHT AELIIPELPA THPPQNRTHW 240VLLGSILLFL IVVSTVLLFL RKQVRMLDVE KCGVEDTSSK NRNDTQFEET 290Macaca mulatta PD-L1 (SEQ ID NO: 18)MRIFAVFIFT IYWHLLNAFT VTVPKDLYVV EYGSNMTIEC RFPVEKQLGL  60TSLIVYWEME DKNIIQFVHG EEDLKVQHSN YRQRAQLLKD QLSLGNAALR 120ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE 180HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL LNVTSTLRIN 240TTANEIFYCI FRRLGPEENH TAELVIPELP LALPPNERTH LVILGAIFLL 300LGVALTFIFY LRKGRMMDMK KSGIRVTNSK KQRDTQLEET 340

B7-H1-Ig proteins are described in WO/2001/014557 (pub. 1 Mar. 2001) andin WO/2002/079499 (pub. 10 Oct. 2002).

3. PD-1 and Other Polypeptides

Other useful polypeptides of the invention include those that bind tothe ligands of the PD-1 receptor. These include the PD-1 receptorprotein, or soluble fragments thereof, which can bind to the PD-1ligands, such as B7-H1 or B7-DC, and prevent binding to the endogenousPD-1 receptor, thereby preventing inhibitory signal transduction. B7-H1has also been shown to bind the protein B7.1 (Butte et al., Immunity,Vol. 27, pp. 111-122, (2007)). Such fragments also include the solubleECD portion of the PD-1 protein that includes mutations, such as theA99L mutation, that increases binding to the natural ligands (Molnar etal., Crystal structure of the complex between programmed death-1 (PD-1)and its ligand PD-L2, PNAS, Vol. 105, pp. 10483-10488 (29 Jul. 2008)).B7-1 or soluble fragments thereof, which can bind to the B7-H1 ligandand prevent binding to the endogenous PD-1 receptor, thereby preventinginhibitory signal transduction, are also useful.

PD-1 polypeptides useful in the methods of the invention are as follows:

Human PD-1 (SEQ ID NO: 19)MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFFPA LLVVTEGDNA TFTCSFSNTS  60ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT 120YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS 180LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP 240CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL 288Cynomolgus monkey PD-1 (SEQ ID NO: 20)MQIPQAPWPV VWAVLQLGWR PGWFLESPDR PWNAPTFSPA LLLVTEGDNA TFTCSFSNAS  60ESPVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTRL PNGRDFHMSV VRARRNDSGT 120YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS 180LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP 240CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL 288

In accordance with the invention, because B7-1 and fragments thereof canalso bind to B7-H1 and send inhibitory transduction to T cells throughB7-H1, blocking of this interaction can also reduce inhibitory signaltransduction that occurs through B7-H1. Compounds for use in theinvention include those molecules that block this type of interaction.Such molecules have been disclosed in Butte et al (2007), supra, andinclude anti-B7-H1 antibodies with dual-specificity that block eitherthe B7-H1:B7-1 and B7-H1:PD-1 interaction as well as antibodiesexhibiting mono-specificity that block the PD-L1:B7-1 interaction.Compounds that block this interaction by blocking B7-1 are also useful,and include anti-B7-1 antibodies.

4. Variant Polypeptides

Polypeptides useful in the invention, as described, include those thatare mutated to contain one or more amino acid substitutions, deletions,or insertions. Methods for mutagenesis are known in the art. The mutatedor variant polypeptides inhibit or reduce inhibitory signal transductionthrough PD-1 receptors by binding to ligands of PD-1. Alternatively, thevariants (e.g. B7-DC polypeptides) can bind to the PD-1 receptor andinhibit, reduce, or block inhibitory signal transduction through thePD-1 receptor. The variant polypeptides may be of any species of origin.In one embodiment, the variant polypeptide is from a mammalian species.In a preferred embodiment, the variant polypeptide is of murine orprimate, preferably human, origin.

In one embodiment the variant polypeptide is a B7-DC polypeptide thathas the same binding affinity to PD-1 as wildtype or non-variant B7-DCbut does not have or has less than 10% ability to trigger inhibitorysignal transduction through the PD-1 receptor relative to a non-mutatedB7-DC polypeptide. In other embodiments, the variant B7-DC polypeptidehas 10%, 20%, 30%, 40%, 50%, or 60% more binding affinity to PD-1 thanwildtype B7-DC without triggering PD-1 inhibitory signalingtransduction.

A variant polypeptide (e.g. a variant B7-DC polypeptide) includes thosehaving any combination of amino acid substitutions, deletions orinsertions so long as the PD-1 antagonizing activity is notsubstantially reduced versus the wild type. However, where there is sucha reduction, this should be by no more than half that of the wild typeso that said variant has at least 50% of the PD-1 antagonist activity ofthe wild type protein, preferably at least 60%, more preferably at least80%, most preferably at least 90% or 95%, with at least 100% beingespecially preferred. Increases in such activity resulting from saidvariant is even more desirable. In one embodiment, isolated B7-DCvariant polypeptides have amino acid alterations such that their aminoacid sequence shares at least 60, 70, 80, 85, 90, 95, 97, 98, 99, 99.5or 100% identity with an amino acid sequence of a wild type B7-DCpolypeptide, especially that from a mammal, preferably wild type murineor wild type primate, preferably human, B7-DC polypeptide.

Polypeptide sequence identity can be calculated using the definition of% identity provided hereinabove.

Amino acid substitutions in polypeptides may be “conservative” or“non-conservative”.

B7 family molecules, including B7-DC, are expressed at the cell surfacewith a membrane proximal constant IgC domain and a membrane distal IgVdomain. Receptors for these ligands share a common extracellularIgV-like domain. Interactions of receptor-ligand pairs are mediatedpredominantly through residues in the IgV domains of the ligands andreceptors. In general, IgV domains are described as having two sheetsthat each contains a layer of β-strands. These β-strands are referred toas A′, B, C, C′, C″, D, E, F and G. In one embodiment the B7-DC variantpolypeptides contain amino acid alterations (i.e., substitutions,deletions or insertions) within one or more of these β-strands in anypossible combination. In another embodiment, B7-DC variants contain oneor more amino acid alterations (i.e., substitutions, deletions orinsertions) within the A′, C, C′, C″, D, E, F or G β-strands. In oneembodiment, B7-DC variants contain one or more amino acid alterations inthe G β-strand.

With respect to murine or primate, preferably human, B7-DC, a variantB7-DC polypeptide can contain, without limitation, substitutions,deletions or insertions at positions that do not substantially reducebinding to PD-1 relative to non-mutated B7-DC.

It is understood, however, that substitutions at the recited amino acidpositions can be made using any amino acid or amino acid analog. Forexample, the substitutions at the recited positions can be made with anyof the naturally-occurring amino acids (e.g., alanine, aspartic acid,asparagine, arginine, cysteine, glycine, glutamic acid, glutamine,histidine, leucine, valine, isoleucine, lysine, methionine, proline,threonine, serine, phenylalanine, tryptophan, or tyrosine).

While the substitutions described herein are with respect to mouse andprimate, especially human, B7-DC, it is noted that one of ordinary skillin the art could readily make equivalent alterations in thecorresponding polypeptides from other species (e.g., rat, hamster,guinea pig, gerbil, rabbit, dog, cat, horse, pig, sheep, cow ornon-human primate).

Preferred fragments include all or part of the extracellular domain ofB7-DC effective to bind to PD-1.

In one embodiment, variant B7-DC polypeptide fragments are those thatretain the ability to bind to PD-1 without triggering PD-1 inhibitorysignal transduction. One embodiment provides a variant B7-DC polypeptidethat is a fragment of full-length B7-DC and typically has at least 20percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80percent, 90 percent, 95 percent, 98 percent, 99 percent, 100 percent, oreven more than 100 percent of the PD-1 antagonist activity of thefull-length variant B7-DC polypeptide.

Useful fragments of variant B7-DC polypeptides include solublefragments. Soluble B7-DC fragments are fragments of B7-DC that may beshed, secreted or otherwise extracted from the producing cells. In oneembodiment, variant B7-DC polypeptide fragments include the entireextracellular domain of B7-DC. The extracellular domain of B7-DCincludes amino acids from about 20 to about amino acid 221 of murine orprimate, preferably human, B7-DC. In another embodiment, variant B7-DCpolypeptide fragments include the IgC and IgV domains of B7-DC. Inanother embodiment, variant B7-DC polypeptide fragments include the IgVdomain of B7-DC.

In one embodiment, variant B7-DC polypeptide fragments contain a regionof the polypeptide that is important for binding affinity for PD-1.These polypeptide fragments are useful to bind to and block the PD-1receptor to prevent native ligands from binding to PD-1 receptor,thereby enhancing an immune response. Inhibiting interactions of nativeB7-H1 or B7-DC with PD-1 inhibits the suppression of immune responsesthat would otherwise occur. A polypeptide fragment of mouse or primate,preferably human, B7-DC that binds to PD-1 contains, by way ofnon-limiting example, amino acids 101-105, or 111-113. The binding ofB7-H1 to PD-1 receptor typically is inhibited by at least 50 percent, orby at least 60 percent, or by at least 70 percent, or by at least 75percent, or by at least 80 percent, or by at least 90 percent, or by atleast 95 percent, or more compared to the level of binding of B7-H1 toPD-1 in the absence of the fragment.

Human PD-1 mutant A99L binds B7-DC and B7-H1 with higher affinity thanunmutated human PD-1 (Lazar Molnar et al PNAS 105 p. 10483-10488(2008)). In one embodiment of the invention, the compound acting toreduce inhibitory signal transduction is a soluble protein, such as theECD of PD-1 incorporating this mutation.

5. Modified Polypeptides

Polypeptides useful in the invention, as described, including variants,homologs and fragments thereof, can be modified by chemical moietiesfound associated with polypeptides in the normal cellular environment,for example, by phosphorylation, methylation, amidation, sulfation,acylation, glycosylation, sumoylation and ubiquitylation of thepolypeptide.

Such polypeptides may also be modified by chemical moieties that are notnormally part of polypeptides in a cellular environment. Suchmodifications can be introduced into the molecule by reacting targetedamino acid residues of the polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or terminalresidues. Another useful modification is cyclization of the protein.Such modifications also include introduction of a label capable ofproviding a detectable signal, either directly or indirectly, including,but not limited to, radioisotopes and fluorescent compounds.

Examples of chemical derivatives of the polypeptides include lysinyl andamino terminal residues derivatized with succinic or other carboxylicacid anhydrides. Derivatization with a cyclic carboxylic anhydride hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate. Carboxyl side groups, aspartyl or glutamyl, may beselectively modified by reaction with carbodiimides (R—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues can be converted to asparaginyl andglutaminyl residues by reaction with ammonia. Polypeptides of theinvention can also include one or more D-amino acids that aresubstituted for one or more L-amino acids.

In other embodiments, the potentiating agent, such as CTX, may be itselfpart of the compound that reduces inhibitory signal transduction, suchas where the potentiating agent is chemically linked to a PD-1antagonist of the invention.

6. Fusion Proteins

Fusion polypeptides having a first fusion partner, or polypeptideportion, comprising all or a part of a PD-1 antagonist protein, a B7-DCpolypeptide for example, (including variants, homologs and fragmentsthereof) fused (i) directly to a second polypeptide or, (ii) optionally,fused to a linker peptide sequence that is fused to the secondpolypeptide are also provided. The presence of the fusion partner canalter, for example, the solubility, affinity and/or valency of the PD-1antagonist polypeptide. The disclosed fusion proteins include anycombination of amino acid alteration (i.e., substitution, deletion orinsertion), fragment, and/or modification of a PD-1 antagonistpolypeptide as described above. In one embodiment, B7-DC fusion proteinsinclude the extracellular domain of a B7-DC protein as the first bindingpartner. In another embodiment, such B7-DC fusion proteins include theIgV and IgC domain of a B7-DC protein as the first binding partner. Inanother embodiment, variant B7-DC fusion proteins include the IgV domainof a B7-DC protein as the first binding partner.

Representative first fusion partners include primate, preferably human,or murine B7-DC polypeptide, fragments thereof, and variants thereofdisclosed hereinabove. Preferred fragments include the extracellulardomain of B7-DC. As recited, the extracellular domain can include 1-10contiguous amino acids of a signal peptide, B7-DC transmembrane domain,or both.

In one embodiment, the compositions and/or products and/or methods ofthe invention utilize PD-1 receptor antagonist, especially polypeptides,including variants, homologs and fragments thereof, that are coupled toother polypeptides to form fusion proteins that antagonize the PD-1receptor by binding a PD-1 ligand, such as B7-H1, thereby inhibiting theligand from interacting with PD-1. In another embodiment, PD-1 receptorantagonist polypeptides, or variants thereof, are coupled to otherpolypeptides to form fusion proteins that antagonize the PD-1 receptorby binding to and blocking the PD-1 receptor and inhibit or reduceinhibitory signal transduction through PD-1.

The second polypeptide binding partner, or second polypeptide portion,may be N-terminal or C-terminal relative to the PD-1 antagonistpolypeptide. In a preferred embodiment, the second polypeptide isC-terminal to the PD-1 antagonist polypeptide.

In a preferred embodiment, the fusion protein contemplated for use inthe methods and compositions and/or products of the invention comprisesat least a portion of an antibody. With the advent of methods ofmolecular biology and recombinant technology, it is now possible toproduce antibody molecules by recombinant means and thereby generategene sequences that code for specific amino acid sequences found in thepolypeptide structure of the antibodies. Such antibodies can be producedby either cloning the gene sequences encoding the polypeptide chains ofsaid antibodies or by direct synthesis of said polypeptide chains, within vitro assembly of the synthesized chains to form active tetrameric(H₂L₂) structures with affinity for specific epitopes and antigenicdeterminants. This has permitted the ready production of antibodieshaving sequences characteristic of neutralizing antibodies fromdifferent species and sources.

Regardless of the source of the antibodies, or how they arerecombinantly constructed, or how they are synthesized, in vitro or invivo, using transgenic animals, such as cows, goats and sheep, usinglarge cell cultures of laboratory or commercial size, in bioreactors orby direct chemical synthesis employing no living organisms at any stageof the process, all antibodies have a similar overall 3 dimensionalstructure. This structure is often given as H₂L₂ and refers to the factthat antibodies commonly comprise 2 light (L) amino acid chains and 2heavy (H) amino acid chains. Both chains have regions capable ofinteracting with a structurally complementary antigenic target. Theregions interacting with the target are referred to as “variable” or “V”regions and are characterized by differences in amino acid sequence fromantibodies of different antigenic specificity.

In preferred embodiments, the PD-1 receptor antagonist polypeptides,including fragments, mutants and other variants, have a first fusionpartner having all or a part of a B7-DC protein or variant thereof fused(i) directly to a second polypeptide or, (ii) optionally, fused to alinker peptide sequence that is fused to the second polypeptide. Thepresence of the fusion partner can alter the solubility, affinity and/orvalency of the B7-DC polypeptide. In more preferred embodiments, B7-DCpolypeptides are fused to one or more domains of an Ig heavy chainconstant region, more preferably an amino acid sequence corresponding tothe hinge, C_(H)2 and C_(H)3 regions of a human immunoglobulin Cγ1 chainor to the hinge, C_(H)2 and C_(H)3 regions of a murine immunoglobulinCγ2a chain. In a preferred embodiment, the constant region preferablyincludes a mutation (for example N297Q) to eliminate or reduce Fcreceptor binding.

The hinge, C_(H)2 and C_(H)3 regions of a human immunoglobulin Cγ1 chainhas the following amino acid sequence:

(SEQ ID NO: 6)EPKSCDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMTSR TPEVTCVVVD VSHEDPEVKF  60NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT 120ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 180PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK 232.

The hinge, C_(H)2 and C_(H)3 regions of a murine immunoglobulin Cγ2achain has the following amino acid sequence:

(SEQ ID NO: 7)EPRGPTIKPC PPCKCPAPNL LGGPSVFIFP PKIKDVLMIS LSPTVTCVVV DVSEDDPDVQ  60ISWFVNNVEV HTAQTQTHRE DYNSTLRVVS ALPIQHQDWM SGKEFKCKVN NKDLPAPIER 120TISKPKGSVR APQVYVLPPP EEEMTKKQVT LTCMVTDFMP EDIYVEWTNN GKTELNYKNT 180EPVLDSDGSY FMYSKLRVEK KNWVERNSYS CSVVHEGLHN HHTTKSFSRT PGK 233

Exemplary murine B7-DC fusion proteins contain amino acids 20-221 ofmurine B7-DC fused to amino acids 237-469 of murine IgG2a (CAA49868).Human B7-DC fusion proteins can contain amino acids 20-221 of humanB7-DC fused to amino acids 245-476 of human IgG1 (AAA02914). The signalpeptides for B7-DC fusion proteins can be the endogenous signal peptidesor any other signal peptide that facilitates secretion of the fusionprotein from a host.

A representative murine B7-DC-Ig fusion protein is encoded by thenucleic acid sequence of SEQ ID NO:8.

It will be appreciated that the disclosed nucleic acid sequences can becodon-optimized to increase levels of expression for synthesizing thefusion proteins useful in the methods and compositions of the presentinvention. Methods for codon optimization are known in the art.

The murine B7-DC-Ig fusion protein encoded by SEQ ID NO:8 has thefollowing amino acid sequence:

(SEQ ID NO: 9)MLLLLPILNL SLQLHPVAAL FTVTAPKEVY TVDVGSSVSL ECDFDRRECT ELEGIRASLQ  60KVENDTSLQS ERATLLEEQL PLGKALFHIP SVQVRDSGQY RCLVICGAAW DYKYLTVKVK 120ASYMRIDTRI LEVPGTGEVQ LTCQARGYPL AEVSWQNVSV PANTSHIRTP EGLYQVTSVL 180RLKPQPSRNF SCMFWNAHMK ELTSAIIDPL SRMEPKVPRT WEPRGPTIKP CPPCKCPAPN 240LLGGPSVFIF PPKIKDVLMI SLSPIVTCVV VDVSEDDPDV QISWFVNNVE VHTAQTQTHR 300EDYNSTLRVV SALPIQHQDW MSGKEFKCKV NNKDLPAPIE RTISKPKGSV RAPQVYVLPP 360PEEEMTKKQV TLTCMVTDFM PEDIYVEWTN NGKTELNYKN TEPVLDSDGS YFMYSKLRVE 420KKNWVERNSY SCSVVHEGLH NHHTTKSFSR TPGK 454

SEQ ID NO:10 provides the amino acid sequence for murine B7-DC-Ig fusionprotein without the signal sequence.

(SEQ ID NO: 10)LFTVTAPKEV YTVDVGSSVS LECDFDRREC TELEGIRASL QKVENDTSLQ SERATLLEEQ  60LPLGKALFHI PSVQVRDSGQ YRCLVICGAA WDYKYLTVKV KASYMRIDTR ILEVPGTGEV 120QLTCQARGYP LAEVSWQNVS VPANTSHIRT PEGLYQVTSV LRLKPQPSRN FSCMFWNAHM 180KELTSAIIDP LSRMEPKVPR TWEPRGPTIK PCPPCKCPAP NLLGGPSVFI FPPKIKDVLM 240ISLSPIVTCV VVDVSEDDPD VQISWFVNNV EVHTAQTQTH REDYNSTLRV VSALPIQHQD 300WMSGKEFKCK VNNKDLPAPI ERTISKPKGS VRAPQVYVLP PPEEEMTKKQ VTLTCMVTDF 360MPEDIYVEWT NNGKTELNYK NTEPVLDSDG SYFMYSKLRV EKKNWVERNS YSCSVVHEGL 420HNHHTTKSFS RTPGK 435

In one embodiment human B7-DC-Ig is encoded by the nucleic acid sequenceof SEQ ID NO:11, encoding the amino acid sequence for human B7-DC-Ig:

(SEQ ID NO: 12)MIFLLLMLSL ELQLHQIAAL FTVTVPKELY IIEHGSNVTL ECNFDTGSHV NLGAITASLQ  60KVENDTSPHR ERATLLEEQL PLGKASFHIP QVQVRDEGQY QCIIIYGVAW DYKYLTLKVK 120ASYRKINTHI LKVPETDEVE LTCQATGYPL AEVSWPNVSV PANTSHSRTP EGLYQVTSVL 180RLKPPPGRNF SCVFWNTHVR ELTLASIDLQ SQMEPRTHPT WEPKSCDKTH TCPPCPAPEL 240LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE 300QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS 360RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK 420SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 453

The present invention specifically contemplates embodiments where themature fusion protein useful in the methods and compositions of theinvention have the signal sequence removed. In a preferred embodiment,the signal sequence is completely removed.

SEQ ID NO:13 provides the amino acid sequence for human B7-DC-Ig withoutthe signal sequence.

(SEQ ID NO: 13)LFTVTVPKEL YIIEHGSNVT LECNFDTGSH VNLGAITASL QKVENDTSPH RERATLLEEQ  60LPLGKASFHI PQVQVRDEGQ YQCIIIYGVA WDYKYLTLKV KASYRKINTH ILKVPETDEV 120ELTCQATGYP LAEVSWPNVS VPANTSHSRT PEGLYQVTSV LRLKPPPGRN FSCVFWNTHV 180RELTLASIDL QSQMEPRTHP TWEPKSCDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI 240SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 300LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV SLTCLVKGFY 360PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH 420NHYTQKSLSL SPGK 434.

The present invention specifically contemplates embodiments where thedisclosed B7-DC-Ig fusion proteins used in the methods and compositionsdisclosed herein have at least about 80%, 85%, 90%, 99% or 100% sequenceidentity to SEQ ID NO: 9, 10, 12, or 13.

In another embodiment of the invention, the fusion polypeptide may havebi-specific function whereby the first fusion partner binds to a ligandof PD-1, such as B7-H1, and the second fusion partner binds to the PD-1receptor without triggering inhibitory signal transduction through thePD-1 receptor.

While a polypeptide useful in the invention may be monomeric or dimeric,the fusion proteins themselves may be present in a monomeric or anoligomeric form, preferably as a dimer. In specific embodiments, thefusion proteins useful as PD-1 antagonists in the methods andcompositions of the invention may assemble spontaneously intooligomeric, especially dimeric, forms or may be chemically linked toform such oligomers by means well known in the art. For example, afusion protein useful in practicing the invention may itself comprise aportion of a B7-DC polypeptide fused to a portion of an antibody andthese may be further assembled into a dimer. In one such example, apolypeptide for use in the invention is fused as a single amino acidchain to the Fc region of an antibody (such as where this construct isexpressed from a single recombinant polynucleotide), after which twosuch fusion products are linked to each other to form a homodimer, suchas by a disulfide linkage between the respective Fc regions.

Such dimeric products may be homodimers (where both monomeric fusionproteins are identical) or may be heterodimers (where two differentfusion proteins are linked to each other). The individual monomers ofsuch dimers may be linked by any means known in the art, such as bycovalent linkage (e.g., a disulfide bond) or by non-covalent linkage(such as an ionic interaction). The B7-DC-Ig used in the examples of theinvention were present in the form of a homodimer having 2 copies of SEQID NO: 10 linked together by a disulfide linkage. In addition, theheterodimers of the invention include bispecific proteins and fusionproteins wherein one monomeric portion binds to PD-1 and the other bindsto a natural ligand of PD-1. Such heterodimers are formed by coupling ofpolypeptides and fusion proteins fully described elsewhere herein.

In another useful embodiment of the invention, the PD-1 antagonist is aheterodimer, such as where two fusion proteins are linked together butthey are not of identical amino acid sequence. In a specific example,each monomer may comprise an Fc portion of an antibody linked to anactive fragment of a B7-DC polypeptide where these active fragments arefrom different portions of the B7-DC polypeptide or where a fusionprotein comprising an Fc portion of an antibody fused to a full lengthnative B7-DC polypeptide is linked (for example, cross-linked) to afusion protein comprising an Fc portion of an antibody and an activefragment of a full length native B7-DC polypeptide. In each such case,the portion of the antibody used in forming each monomeric fusionprotein may be different between the two monomeric units. Any suchdimeric combination is specifically contemplated by the methods andcompositions of the invention.

In a preferred dimeric fusion protein, the dimer results from thecovalent bonding of Cys residue in the CH regions of two of the Ig heavychains that are the same Cys residues that are disulfide linked indimerized normal Ig heavy chains.

Still another embodiment provides a tetramer construct having a BirAsubstrate fused to the extracellular domain of a variant B7-DCpolypeptide. Methods for making tetramer constructs are known in the art(see Pertovas, et al., J. Exp. Med., 203:2281 (2006)).

7. Anti-PD-1 and Other Antibodies

Other PD-1 antagonists contemplated by the methods of this inventioninclude antibodies that bind to PD-1 or ligands of PD-1, and otherantibodies.

In one aspect, the present invention relates to a method of increasing aT cell response in a mammal in need thereof, comprising administering tosaid mammal an effective treatment regimen comprising an anti-PD-1antibody and a potentiating agent, wherein said treatment regimen iseffective to increase the T cell response of said mammal to saidantigen.

Anti-PD-1 antibodies useful in the treatment regimens(s) of theinvention include, but are not limited to, those described in thefollowing publications:

-   PCT/IL03/00425 (Hardy et al., WO/2003/099196)-   PCT/JP2006/309606 (Korman et al., WO/2006/121168)-   PCT/US2008/008925 (Li et al., WO/2009/014708)-   PCT/JP03/08420 (Honjo et al., WO/2004/004771)-   PCT/JP04/00549 (Honjo et al., WO/2004/072286)-   PCT/IB2003/006304 (Collins et al., WO/2004/056875)-   PCT/US2007/088851 (Ahmed et al., WO/2008/083174)-   PCT/US2006/026046 (Korman et al., WO/2007/005874)-   PCT/US2008/084923 (Terrett et al., WO/2009/073533)-   Berger et al., Clin. Cancer Res., Vol. 14, pp. 30443051 (2008).

A specific example of an anti-PD-1 antibody useful in the methods of theinvention is MDX-1106 (see Kosak, US 20070166281 (pub. 19 Jul. 2007) atpar. 42), a human anti-PD-1 antibody, preferably administered at a doseof 3 mg/kg.

In another aspect, the present invention relates to a method ofincreasing a T cell response in a mammal in need thereof, comprisingadministering to said mammal an effective treatment regimen comprisingan anti-PD-1 ligand antibody, an anti-B7-H1 antibody for example, and apotentiating agent, wherein said treatment regimen is effective toincrease the T cell response of said mammal to said antigen.

Anti-B7-H1 antibodies useful in the treatment regimens(s) of theinvention include, but are not limited to, those described in thefollowing publications:

-   PCT/US06/022423 (WO/2006/133396, pub. 14 Dec. 2006)-   PCT/US07/088851 (WO/2008/083174, pub. 10 Jul. 2008)-   US 2006/0110383 (pub. 25 May 2006)

A specific example of an anti-B7-H1 antibody useful in the methods ofthe invention is MDX-1105 (WO/2007/005874, published 11 Jan. 2007)), ahuman anti-B7-H1 antibody.

For anti-B7-DC antibodies see U.S. Pat. Nos. 7,411,051, 7,052,694,7,390,888, 20060099203

Another embodiment of the invention includes a bi-specific antibody thatcomprises an antibody that binds to the PD-1 receptor bridged to anantibody that binds to a ligand of PD-1, such as B7-H1. In a preferredembodiment, the PD-1 binding portion reduces or inhibits signaltransduction through the PD-1 receptor.

The antibody for use in the invention need not be an anti-PD-1 oranti-PD-1 ligand antibody but may be another antibody useful inmediating the effects of T cells in an immune response. In this aspect,the present invention relates to a method of increasing a T cellresponse to an antigen in a mammal in need thereof, comprisingadministering to said mammal an effective treatment regimen comprisingan anti-CTLA4 antibody and a potentiating agent, wherein said treatmentregimen is effective to increase the T cell response of said mammal tosaid antigen. An example of an anti-CTLA4 antibody contemplated for usein the methods of the invention includes an antibody as described inPCT/US2006/043690 (Fischkoff et al., WO/2007/056539).

Specific examples of an anti-CTLA4 antibody useful in the methods of theinvention are Ipilimumab, also known as MDX-010 or MDX-101, a humananti-CTLA4 antibody, preferably administered at a dose of 10 mg/kg, andTremelimumab a human anti-CTLA4 antibody, preferably administered at adose of 15 mg/kg.

8. Small Molecule PD-1 Antagonists

The PD-1 receptor antagonists can also be small molecule antagonists.The term “small molecule” refers to small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons,preferably between 100 and 2000, more preferably between about 100 andabout 1250, more preferably between about 100 and about 1000, morepreferably between about 100 and about 750, more preferably betweenabout 200 and about 500 daltons. The small molecules often includecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more functional groups.The small molecule antagonists reduce or interfere with PD-1 receptorsignal transduction by binding to ligands of PD-1 such as B7-H1 andB7-DC and preventing the ligand from interacting with PD-1 or by bindingdirectly to and blocking the PD-1 receptor without triggering signaltransduction through the PD-1 receptor.

In one embodiment, such a small molecule may be administered incombination with another PD-1 antagonist or CTLA4 antagonist, such as anantibody specific for PD-1 or one of its ligands or an antibody specificfor CTLA4 or one of its ligands. Thus, such small molecules may beadministered as compounds in one or more of the methods of the inventionor may be administered in combination with other compounds useful in themethods of the invention. For example, a series of small organiccompounds have been shown to bind to the B7-1 ligand to prevent bindingto CTLA4 (see Erbe et al., J. Biol. Chem., Vol. 277, pp. 7363-7368(2002). Such small organics could be administered alone or together withan anti-CTLA4 antibody, in combination with CTX administration, toreduce inhibitory signal transduction of T cells.

In one embodiment, PD-1 antagonists or CTLA4 antagonists contemplatedfor use in the methods of the invention include anti-sense nucleicacids, both DNA and RNA, as well as siRNA molecules. Such anti-sensemolecules prevent expression of PD-1 on T cells as well as production ofT cell ligands, such as B7-H1, PD-L1 and PD-L2. For example, siRNA (forexample, of about 21 nucleotides in length, which is specific for thegene encoding PD-1, or encoding a PD-1 ligand, and whicholigonucleotides can be readily purchased commercially) complexed withcarriers, such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin.Invest. 119(8): 2231-2244 (2009), are readily taken up by cells thatexpress PD-1 as well as ligands of PD-1 and reduce expression of thesereceptors and ligands to achieve a decrease in inhibitory signaltransduction in T cells, thereby activating T cells.

B. Potentiating Agents

In accordance with the invention, the activity of the PD-1 antagonist isincreased, preferably synergistically, by the presence of a potentiatingagent. The potentiating agent acts to increase the efficacy of the PD-1receptor antagonist, possibly by more than one mechanism, although theprecise mechanism of action is not essential to the broad practice ofthe present invention.

In the preferred embodiment, the potentiating agent is cyclophosphamide.Cyclophosphamide (CTX, Cytoxan®, or Neosar®) is an oxazahosphorine drugand analogs include ifosfamide (IFO, Ifex), perfosfamide, trophosphamide(trofosfamide; Ixoten), and pharmaceutically acceptable salts, solvates,prodrugs and metabolites thereof (US patent application 20070202077which is incorporated in its entirety). Ifosfamide (MITOXANA®) is astructural analog of cyclophosphamide and its mechanism of action isconsidered to be identical or substantially similar to that ofcyclophosphamide. Perfosfamide (4-hydroperoxycyclophosphamide) andtrophosphamide are also alkylating agents, which are structurallyrelated to cyclophosphamide. For example, perfosfamide alkylates DNA,thereby inhibiting DNA replication and RNA and protein synthesis. Newoxazaphosphorines derivatives have been designed and evaluated with anattempt to improve the selectivity and response with reduced hosttoxicity (Liang J, Huang M, Duan W, Yu X Q, Zhou S. Design of newoxazaphosphorine anticancer drugs. Curr Pharm Des. 2007; 13(9):963-78.Review). These include mafosfamide (NSC 345842), glufosfamide (D19575,beta-D-glucosylisophosphoramide mustard), S-(−)-bromofosfamide (CBM-11),NSC 612567 (aldophosphamide perhydrothiazine) and NSC 613060(aldophosphamide thiazolidine). Mafosfamide is an oxazaphosphorineanalog that is a chemically stable 4-thioethane sulfonic acid salt of4-hydroxy-CPA. Glufosfamide is IFO derivative in which theisophosphoramide mustard, the alkylating metabolite of IFO, isglycosidically linked to a beta-D-glucose molecule. Additionalcyclophosphamide analogs are described in U.S. Pat. No. 5,190,929entitled “Cyclophosphamide analogs useful as anti-tumor agents” which isincorporated herein by reference in its entirety.

In other embodiments, the potentiating agent is an agent that reducesactivity and/or number of regulatory T lymphocytes (T-regs), preferablySunitinib (SUTENT®), anti-TGFβ or Imatinib (GLEEVAC®). The recitedtreatment regimen may also include administering an adjuvant.

Useful potentiating agents also include mitosis inhibitors, such aspaclitaxol, aromatase inhibitors (e.g. Letrozole) and angiogenesisinhibitors (VEGF inhibitors e.g. Avastin, VEGF-Trap) (see, for example,Li et al., Vascular endothelial growth factor blockade reducesintratumoral regulatory T cells and enhances the efficacy of aGM-CSF-secreting cancer immunotherapy. Clin Cancer Res. 2006 Nov. 15;12(22):6808-16), anthracyclines, oxaliplatin, doxorubicin, TLR4antagonists, and IL-18 antagonists.

C. Pharmaceutical Compositions

In one aspect, the invention relates to a therapeutic composition,comprising a molecule that prevents inhibitory signal transductionthrough PD-1, or a CTLA4 antagonist, and a potentiating agent in apharmaceutically acceptable carrier. The components of said compositionare present in an amount effective to increase a T cell response in amammal. In specific embodiments, the potentiating agent iscyclophosphamide or an analog of cyclophosphamide, examples of suchanalogs having been recited above.

In other specific examples, the potentiating agent is an agent thatreduces activity of regulatory T lymphocytes (T-regs), preferably wherethe activity is reduced due to a decrease in the number of said T-regs.In preferred non-limiting embodiments, the agent is Sunitinib (SUTENT®),anti-TGFβ or Imatinib (GLEEVAC®).

The potentiating agent useful in formulating compositions of theinvention also include mitosis inhibitors, such as paclitaxol, aromataseinhibitors (e.g. Letrozole), angiogenesis inhibitors (VEGF inhibitorse.g. Avastin, VEGF-Trap), anthracyclines, oxaliplatin, doxorubicin, TLR4antagonists, and IL-18 antagonists.

A therapeutic composition of the invention also optionally comprises atleast one additional agent that may be one or more of an anti-PD-1antibody, an anti-CTLA4 antibody, a mitosis inhibitor, an aromataseinhibitor, an A2a adenosine receptor (A2AR) antagonist, or anangiogenesis inhibitor.

Any of the therapeutic compositions of the invention may also containone or more adjuvants as described herein.

A PD-1 antagonist useful as a component of a therapeutic composition ofthe invention includes any of the PD-1 antagonists recited herein foruse in any of the methods of the invention. For example, such PD-1antagonist includes any of the fusion proteins recited herein. Suchantagonist can also be any of the polypeptides or PD-1 binding fragmentsrecited herein for use as the first polypeptide portion of any of thefusion proteins described for use in any of the methods of theinvention. Such antagonist can further be an antibody, such as any ofthe known anti-PD-1, -B7-DC or -B7-H1 antibodies mentioned herein.

A therapeutic composition of the invention also includes, in addition toor in place of the aforementioned PD-1 antagonist, an anti-CTLA4antibody. Such a composition would therefore contain such an anti-CTLA4antibody and a potentiating agent of the kind already described herein.

A therapeutic composition of the invention finds use in any of themethods of the invention disclosed herein. Such composition, whileintended for use as an active treatment of a disease condition, may alsofind use as prophylactic compositions to prevent any of the diseasesrecited herein.

In one aspect, the present invention contemplates a therapeuticcomposition comprising a PD-1 antagonist and a potentiating agent in apharmaceutically acceptable carrier, wherein the PD-1 antagonist and thepotentiating agent are together present in an amount effective toincrease a T cell response in a mammal.

Therapeutic compositions within the scope of the invention includecompositions comprising any and all combinations of the PD-1 antagonistsand/or antibodies disclosed herein with any of the recited potentiatingagents. By way of non-limiting examples, a therapeutic composition ofthe invention includes a composition comprising an effective amount ofone or more PD-1 antagonists, such as a combination of any or all of thefull length polypeptides enumerated herein as specific SEQ ID NOs. orhomologs thereof together with one or more fragments of any of saidpolypeptides, including where any or all of these are fused to otherproteins, such as being fused to one or more immunoglobulins recitedherein, or not so fused, and comprising one or more potentiating agents,such as cyclophosphamide alone, or cyclophosphamide plus one or moreanalogs thereof, of just one or more analogs of cyclophosphamide, or thepotentiating agent may consist of cyclophosphamide and an agent thatreduces T reg number in a mammal receiving the composition, or mayconsist of a cyclophosphamide analog plus an agent that reduces T regnumber or the potentiating agent may consist only of one or more agentsthat reduce T reg number or other Treg activity. All such combinationsare contemplated by the invention so long as the composition comprisesat least one PD-1 antagonist and/or antibody mediating T cell activityand at least one potentiating agent.

The compositions of the invention may also include additional activeagents. In preferred embodiments of any of the compositions of theinvention, the pharmaceutical or therapeutic composition furthercomprises at least one additional agent selected from the groupconsisting of an anti-PD-1 antibody, an anti-CTLA4 antibody, a mitosisinhibitor, such as paclitaxel, an aromatase inhibitor, such asletrozole, an A2AR antagonist, an angiogenesis inhibitor,anthracyclines, oxaliplatin, doxorubicin, TLR4 antagonists, and IL-18antagonists.

The PD-1 antagonist and/or potentiating agent may be administered by anysuitable means. In a preferred embodiment, the PD-1 antagonist and/orpotentiating agent is administered in an aqueous solution, by parenteralinjection. The formulation may also be in the form of a suspension oremulsion. In general, pharmaceutical compositions are provided includingeffective amounts of a peptide or polypeptide, and optionally includepharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions includediluents sterile water, buffered saline of various buffer content (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength; and optionally,additives such as detergents and solubilizing agents (e.g., TWEEN 20,TWEEN 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol). Examples of non-aqueoussolvents or vehicles are propylene glycol, polyethylene glycol,vegetable oils, such as olive oil and corn oil, gelatin, and injectableorganic esters such as ethyl oleate. The formulations may be lyophilizedand redissolved/resuspended immediately before use. The formulation maybe sterilized by, for example, filtration through a bacteria retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions.

Pharmaceutical compositions of the invention may be administered byparenteral (intramuscular, intraperitoneal, intravenous (IV) orsubcutaneous injection), transdermal (either passively or usingiontophoresis or electroporation), or transmucosal (nasal, vaginal,rectal, or sublingual) routes of administration. The methods of theinvention do not preclude administering the PD-1 antagonist and thepotentiating agent by separate and different routes (e.g. topically).

The PD-1 antagonist and the potentiating agent may be administered atthe same time, or at different times, with the potentiating agent beingadministered before or after the PD-1 antagonist. In one embodiment, apotentiating agent is administered both before and after the PD-1antagonist. In one such embodiment, the same potentiating agent isadministered before and after the PD-1 antagonist. In anotherembodiment, the potentiating agent administered before the PD-1antagonist.

As used herein the term “effective amount” or “therapeutically effectiveamount” means a dosage sufficient to treat, inhibit, or alleviate one ormore symptoms of the disorder being treated or to otherwise provide adesired pharmacologic and/or physiologic effect. The precise dosage willvary according to a variety of factors such as subject-dependentvariables (e.g., age, immune system health, etc.), the disease, and thetreatment being effected. Therapeutically effective amounts of PD-1receptor antagonists and/or antibodies together with a potentiatingagents cause an immune response to be activated or sustained.

The selected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment desired.Generally dosage levels of 0.001 to 50 mg/kg of body weight daily areadministered to mammals. Preferrably, said dose is 1 to 50 mg/kg, morepreferably 1 to 40 mg/kg, or even 1 to 30 mg/kg, with a dose of 2 to 20mg/kg being also a preferred dose. Examples of other dosages include 2to 15 mg/kg, or 2 to 10 mg/kg or even 3 to 5 mg/kg, with a dose of about4 mg/kg being a specific example.

For treatment regimens using a potentiating agent and an antibody, suchas an anti-PD-1 antibody or an anti-CTLA4 antibody, dosages are commonlyin the range of 0.1 to 100 mg/kg, with shorter ranges of 1 to 50 mg/kgpreferred and ranges of 10 to 20 mg/kg being more preferred. Anappropriate dose for a human subject is between 5 and 15 mg/kg, with 10mg/kg of antibody (for example, human anti-PD-1 antibody, like MDX-1106)most preferred (plus a suitable dose of cyclophosphamide or otherpotentiating agent given up to about 24 hours before the antibody).

In general, by way of example only, dosage forms based on body weightfor any of the signal transduction antagonists useful in the methods ofthe invention include doses in the range of 5-300 mg/kg, or 5-290 mg/kg,or 5-280 mg/kg, or 5-270 mg/kg, or 5-260 mg/kg, or 5-250 mg/kg, or 5-240mg/kg, or 5-230 mg/kg, or 5-220 mg/kg, or 5-210 mg/kg, or 20 to 180mg/kg, or 30 to 170 mg/kg, or 40 to 160 mg/kg, or 50 to 150 mg/kg, or 60to 140 mg/kg, or 70 to 130 mg/kg, or 80 to 120 mg/kg, or 90 to 110mg/kg, or 95 to 105 mg/kg, with doses of 3 mg/kg, 5 mg/kg, 7 mg/kg, 10mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 50 mg/kg and 100 mg/kgbeing specific examples of preferred doses. Such doses may, of course,be repeated. The dose will, of course, be correlated with the identityof the mammal receiving said dose. Doses in the above-recited mg/kgranges are convenient for mammals, including rodents, such as mice andrats, and primates, especially humans, with doses of about 5 mg/kg,about 10 mg/kg and about 15 mg/kg being especially preferred fortreating humans.

In accordance with the treatment regimen of the invention, thepotentiating agent, for example cyclophosphamide, is administered innon-toxic doses that vary depending on the animal. In specificembodiments, the potentiating agent is administered by any suitablemeans of administration, including parenteral or oral, the formerincluding system administration, such as intravenous. For example, apotentiating agent like cyclophosphamide is normally administeredorally. Such administration may be at any convenient dosage, dependingon the potentiating agent. The dosage in each case may be based on bodyweight or may be administered as a unit dosage.

While CTX itself is nontoxic, some of its metabolites are cytotoxicalkylating agents that induce DNA crosslinking and, at higher doses,strand breaks. Many cells are resistant to CTX because they express highlevels of the detoxifying enzyme aldehyde dehydrogenase (ALDH). CTXtargets proliferating lymphocytes, as lymphocytes (but not hematopoieticstem cells) express only low levels of ALDH, and cycling cells are mostsensitive to DNA alkylation agents.

Low doses of CTX (<200 mg/kg) can have immune stimulatory effects,including stimulation of anti-tumor immune responses in humans and mousemodels of cancer (Brode & Cooke Crit. Rev. Immunol. 28:109-126 (2008)).These low doses are sub-therapeutic and do not have a direct anti-tumoractivity. In contrast, high doses of CTX inhibit the anti-tumorresponse. Several mechanisms may explain the role of CTX in potentiationof anti-tumor immune response: (a) depletion of CD4+CD25+FoxP3+Treg (andspecifically proliferating Treg, which may be especially suppressive),(b) depletion of B lymphocytes; (c) induction of nitric oxide (NO),resulting in suppression of tumor cell growth; (d) mobilization andexpansion of CD11b+Gr-1+MDSC. These primary effects have numeroussecondary effects; for example following Treg depletion macrophagesproduce more IFN-γ and less IL-10. CTX has also been shown to inducetype I IFN expression and promote homeostatic proliferation oflymphocytes.

Treg depletion is most often cited as the mechanism by which CTXpotentiates the anti-tumor immune response. This conclusion is based inpart by the results of adoptive transfer experiments. In the AB1-HAtumor model, CTX treatment at Day 9 gives a 75% cure rate. Transfer ofpurified Treg at Day 12 almost completely inhibited the CTX response(van der Most et al. Cancer Immunol. Immunother. 58:1219-1228 (2009). Asimilar result was observed in the HHD2 tumor model: adoptive transferof CD4+CD25+Treg after CTX pretreatment eliminated therapeutic responseto vaccine (Taieb, J. J. Immunol. 176:2722-2729 (2006)).

Numerous human clinical trials have demonstrated that low dose CTX is asafe, well-tolerated, and effective agent for promoting anti-tumorimmune responses (Bas, & Mastrangelo Cancer Immunol. Immunother. 47:1-12(1998)).

The optimal dose for CTX to potentiate an anti-tumor immune response, isone that lowers overall T cell counts by lowering Treg levels below thenormal range but is subtherapeutic (see Machiels et al. Cancer Res.61:3689-3697 (2001)).

In human clinical trials where CTX has been used as animmunopotentiating agent, a dose of 300 mg/m² has usually been used. Foran average male (6 ft, 170 pound (78 kg) with a body surface area of1.98 m²), 300 mg/m² is 8 mg/kg, or 624 mg of total protein. In mousemodels of cancer, efficacy has been seen at doses ranging from 15-150mg/kg, which relates to 0.45-4.5 mg of total protein in a 30 g mouse(Machiels et al. Cancer Res. 61:3689-3697 (2001), Hengst et al CancerRes. 41:2163-2167 (1981), Hengst Cancer Res. 40:2135-2141 (1980)).

For larger mammals, such as a primate, preferably human, patient, suchmg/m² doses may be used but unit doses administered over a finite timeinterval may be preferred. Such unit doses may be administered on adaily basis for a finite time period, such as up to 3 days, or up to 5days, or up to 7 days, or up to 10 days, or up to 15 days or up to 20days or up to 25 days, are all specifically contemplated by theinvention. The same regimen may be applied for the other potentiatingagents recited herein.

All such administrations may occur before or after administration of aPD-1 binding molecule of the invention. Alternatively, administration ofone or more doses of a PD-1 binding molecule of the invention may betemprally staggered with the administration of potentiating agent toform a uniform or non-uniform course of treatment whereby one or moredoses of potentiating agent are administered, followed by one or moredoses of a PD-1 binding compound, followed by one or more doses ofpotentiating agent, all according to whatever schedule is selected ordesired by the researcher or clinician administering said agents.

In other specific embodiments, the treatment regimen includes multipleadministrations of one or more PD-1 antagonists. In some embodiments,such multiple administrations of PD-1 antagonists are in conjunctionwith multiple administrations of the same or different potentiatingagents.

As in other embodiments of the invention, here the potentiating agent isadministered at least 1, 2, 3, 5, 10, 15, 20, 24 or 30 hours prior to orafter administering of the PD-1-antagonist.

The pharmaceutical compositions useful herein also contain apharmaceutically acceptable carrier, including any suitable diluent orexcipient, which includes any pharmaceutical agent that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition, and which may be administered without undue toxicity.Pharmaceutically acceptable carriers include, but are not limited to,liquids such as water, saline, glycerol and ethanol, and the like,including carriers useful in forming sprays for nasal and otherrespiratory tract delivery or for delivery to the ophthalmic system. Athorough discussion of pharmaceutically acceptable carriers, diluents,and other excipients is presented in REMINGTON'S PHARMACEUTICAL SCIENCES(Mack Pub. Co., N.J. current edition).

Vaccine compositions (as discussed below) may further incorporateadditional substances to stabilize pH, or to function as adjuvants,wetting agents, or emulsifying agents, which can serve to improve theeffectiveness of the vaccine.

Vaccines are generally formulated for parenteral administration and areinjected either subcutaneously or intramuscularly. Such vaccines canalso be formulated as suppositories or for oral administration, usingmethods known in the art, or for administration through nasal orrespiratory routes.

D. Methods of Manufacture

Isolated PD-1 antagonist polypeptides, including variants, homologs andfragments thereof, either wild-type or mutated, and fusion proteinscomprising any of these, all contemplated for use in the invention, canbe obtained by, for example, chemical synthesis or by recombinantproduction in a host cell. To recombinantly produce a costimulatorypolypeptide, a nucleic acid containing a nucleotide sequence encodingthe polypeptide can be used to transform, transduce, or transfect abacterial or eukaryotic host cell (e.g., an insect, yeast, or mammaliancell). It will be appreciated that the nucleotide sequences can becodon-optimized to increase levels of protein expression in a particularkind of host cell. Methods for codon optimization are well known in theart. In general, nucleic acid constructs include a regulatory sequenceoperably linked to a nucleotide sequence encoding a costimulatorypolypeptide. Regulatory sequences (also referred to herein as expressioncontrol sequences) typically do not encode a gene product, but insteadaffect the expression of the nucleic acid sequences to which they areoperably linked. The signal peptides used to secrete proteins from acell can be the endogenous signal peptides or any other signal peptidethat facilitates secretion of the fusion protein from a host.

For general molecular biology procedures useful in practicing thepresent invention, a number of standard references are available thatcontain procedures well known in the art of molecular biology andgenetic engineering and which procedures need not be further describedherein. Useful references include Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Wuet al, Methods in Gene Biotechnology (CRC Press, New York, N.Y., 1997),and Recombinant Gene Expression Protocols, in Methods in MolecularBiology, Vol. 62, (Tuan, ed., Humana Press, Totowa, N.J., 1997), thedisclosures of which are hereby incorporated by reference.

E. Disease Treatment

Diseases to be treated or prevented by administering a therapeuticcombination provided by the present invention include a malignant tumoror a chronic infectious disease caused by a bacterium, virus, protozoan,helminth, or other microbial pathogen that enters intracellularly. Suchdiseases are often combatted through attack by cytotoxic T lymphocytes.Because the present invention provides combination therapes useful inenhancing T cell responses, through increased T cell activity, increasedT cell proliferation and reduced T cell inhibitory signals, thecombination therapies of the invention have unique advantage in treating(or even preventing) such diseases.

In one embodiment, because viral infections are cleared primarily byT-cells, an increase in T-cell activity is therapeutically useful inenhancing clearance of an infective viral agent from an animal orprimate, preferably human, subject. Thus, the disclosed compounds of theinvention, with PD-1 receptor antagonist activity, together with apotentiating agent work in combination for the treatment of local orsystemic viral infections. Infections that are to be treated by thecompounds of the invention include, but are not limited to,immunodeficiency (e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV),encephalitis, influenza (e.g., human influenza virus A), hepatitis (e.g.HCV, HBV), and common cold (e.g., human rhinovirus) viral infections.Pharmaceutical formulations of PD-1 receptor antagonists compositionscan also be administered to treat systemic viral diseases, including,but not limited to, AIDS, influenza, the common cold, or encephalitis.

Non-viral infections treatable by the compounds of the inventioninclude, but are not limited to, infections cause by microoganismsincluding, but not limited to, Actinomyces, Anabaena, Bacillus,Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter,Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium,Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella,Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B(HIB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, MeningococcusA, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma,Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus,Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella,Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces,Sulfolobus, Thermoplasma, Thiobacillus, Treponema, Vibrio, Yersinia,Cryptococcus neoformans, Histoplasma sp. (such as Histoplasmacapsulatum), Candida albicans, Candida tropicalis, Nocardia asteroides,Rickettsia ricketsii, Rickettsia typhi, Leishmania, Mycoplasmapneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium sp.(such as Plasmodium falciparum), Trypanosoma brucei, Entamoebahistolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosomamansoni.

In one embodiment, the present invention provides methods andcompositions for inducing or enhancing an immune response in host fortreating cancer. The types of cancer that may be treated with theprovided compositions and methods include, but are not limited to, thefollowing: bladder, brain, breast, cervical, colo-rectal, esophageal,kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin,stomach, uterine, ovarian testicular, and hematologic cancer.

Malignant tumors which may be treated are classified herein according tothe embryonic origin of the tissue from which the tumor is derived.Carcinomas are tumors arising from endodermal or ectodermal tissues suchas skin or the epithelial lining of internal organs and glands.Sarcomas, which arise less frequently, are derived from mesodermalconnective tissues such as bone, fat, and cartilage. The leukemias andlymphomas are malignant tumors of hematopoietic cells of the bonemarrow. Leukemias proliferate as single cells, whereas lymphomas tend togrow as tumor masses. Malignant tumors may show up at numerous organs ortissues of the body to establish a cancer.

As a demonstration of the value of the treatment regimens of theinvention, the murine analog of B7-DC-Ig (in which the mouse B7-DC ECD,which shares 72% sequence identity with the human protein, is fused tothe Fc domain of mouse IgG_(2a)) tested in syngeneic mouse tumor modelsfor colon cancer, mastocytoma, and other tumor types incorporating acyclophosphamide (CTX) pre-treatment as described herein.

The results showed that treatment with a single subtherapeutic dose ofCTX, which acts as an immunopotentiating agent, followed by murineB7-DC-IG eradicates established CT26 colon carcinoma tumors in up to 80%of the animals. Further, in CT26 colon carcinoma tumor re-challengestudies, no tumor re-growth was detected in mice that had previouslyeradicated tumor following CTX+murine B7-DC-Ig treatment. These micewere also shown to have an increased tumor-specific CTL populationrelative to naïve mice.

In one embodiment, the present invention contemplates use of a compoundthat reduces inhibitory signal transduction in a T cell, as describedelsewhere herein, in the manufacture of a medicament for increasing a Tcell response by combination therapy wherein said compound isadministered in conjunction with a potentiating agent. Further, thecompound that reduces inhibitory signal transduction in a T cell andsaid potentiating agent are provided as separate medicaments foradministration at different times, preferably where the potentiatingagent is administered prior to the compound that reduces inhibitorysignal transduction, for example, up to 24 hours prior to the inhibitorycompound (or other time intervals recited herein). Preferably, thecompound and potentiating agent are for use in the treatment of aninfectious disease or cancer, including diseases caused by any of theinfectious agents or cancers recited elsewhere herein.

In a preferred embodiment, a compound useful in these methods is arecombinant protein composed of the ECD of human B7-DC fused to the Fcdomain of human IgG₁, referred to herein as B7-DC-Ig.

In one embodiment, the present invention relates to a medical kit foradministering a compound that reduces inhibitory signal transduction ina T cell, as disclosed herein, in combination with a potentiating agent,said kit comprising:

(a) a dosage supply of a compound that reduces inhibitory signaltransduction in a T cell,

(b) a supply of a potentiating agent;

(c) a supply of pharmaceutically acceptable carrier; and

(d) printed instructions for administering the compound in a use asdescribed above.

F. Combination Therapies

Vaccines require strong T cell response to eliminate cancer cells andinfected cells or infectious agents. PD-1 receptor antagonists describedherein can be administered as a component of a vaccine, along with apotentiating agent, to provide a costimulatory signal to T cells.Vaccines disclosed herein include antigens, a PD-1 receptor antagonistand optionally adjuvants and targeting molecules.

The antigens against which the T cell response is enhanced by themethods and composition of the invention includes peptides, proteins,polysaccharides, saccharides, lipids, nucleic acids, or combinationsthereof. The antigens, in the case of disease, are present due to thedisease process.

The disclosed PD-1 receptor antagonists compositions may be administeredin conjunction with prophylactic vaccines, which confer resistance in asubject to subsequent exposure to infectious agents, or in conjunctionwith therapeutic vaccines, which can be used to initiate or enhance asubject's immune response to a pre-existing antigen, such as a tumorantigen in a subject with cancer, or a viral antigen in a subjectinfected with a virus.

The desired outcome of a prophylactic, therapeutic or de-sensitizedimmune response may vary according to the disease, based on principleswell known in the art. For example, an immune response against aninfectious agent may completely prevent colonization and replication ofan infectious agent, affecting “sterile immunity” and the absence of anydisease symptoms. However, a vaccine against infectious agents may beconsidered effective if it reduces the number, severity or duration ofsymptoms; if it reduces the number of individuals in a population withsymptoms; or reduces the transmission of an infectious agent. Similarly,immune responses against cancer, allergens or infectious agents maycompletely treat a disease, may alleviate symptoms, or may be one facetin an overall therapeutic intervention against a disease. For example,the stimulation of an immune response against a cancer may be coupledwith surgical, chemotherapeutic, radiologic, hormonal and otherimmunologic approaches in order to affect treatment.

The methods and products of the invention do not preclude use of anadjuvant in addition to the potentiating agent. Such adjuvant may beadministered, for example, along with the PD-1 antagonist. The adjuvantsuseful in the compositions and methods of the invention include, but arenot limited to, one or more of the following: oil emulsions (e.g.,Freund's adjuvant); saponin formulations; virosomes and viral-likeparticles; bacterial and microbial derivatives; immunostimulatoryoligonucleotides; ADP-ribosylating toxins and detoxified derivatives;alum; BCG; mineral-containing compositions (e.g., mineral salts, such asaluminium salts and calcium salts, hydroxides, phosphates, sulfates,etc.); bioadhesives and/or mucoadhesives; microparticles; liposomes;polyoxyethylene ether and polyoxyethylene ester formulations; muramylpeptides; polyphosphazene; imidazoquinolone compounds; and surfaceactive substances (e.g. lysolecithin, pluronic polyols, polyanions,peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).Useful adjuvants also include immunomodulators such as cytokines,interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.),interferons (e.g., interferon-γ), macrophage colony stimulating factor,and tumor necrosis factor.

Nothing herein precludes the disclosed PD-1 receptor antagonist,including any of the polypeptides, fragments, variants, homologs andfusion proteins disclosed herein, from being administered to a subjectin need thereof in combination with one or more additional therapeuticagents (in addition to the potentiating agent). The additionaltherapeutic agents are selected based on the condition, disorder ordisease to be treated. For example, PD-1 receptor antagonists can beco-administered with one or more additional agents that function toenhance or promote an immune response, and which are considered hereinas active agents.

Such agents include, but are not limited to, amsacrine, bleomycin,busulfan, capecitabine, carboplatin, carmustine, chlorambucil,cisplatin, cladribine, clofarabine, crisantaspase, cytarabine,dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin,epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine,hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin,liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan,mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone,oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine,raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide,teniposide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine,vincristine, vindesine, vinorelbine, or a combination thereof.Representative pro-apoptotic agents include, but are not limited tofludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide,15d-PGJ(2) and combinations thereof.

The therapies provided by the methods and compositions of the presentinvention may also be used in conjunction with other types of therapies,such as radiation treatments, surgery, and the like.

G. Assays for Antagonist Activity

The present invention recites a number specific structures useful inpracticing the methods of the invention. Other compounds possessingantagonist activity and being useful in the methods of the invention mayalso be identified by reference to well known assay procedures foridentifying chemical structures that bind to PD-1, CTLA4, and ligands ofany of these and that also possess the ability to reduce inhibitorysignal transduction in T cells. Some such assays are binding assaysuseful in determining if a selected chemical structure binds toreceptors; these are well known in the art and need not be discussed indetail herein (see, for example, U.S. 2008/0274490, (pub. 6 Nov. 2008)and U.S. Pat. No. 7,105,328 (issued 12 Sep. 2006), each showing assaysfor PD-1 signaling modulators using T cells) the disclosures of whichare hereby incorporated by reference in its entirety. Other assays areused to determine the effects of agents of the invention, such as activefragments, to activate T cells by increasing proliferation and/orproduction of cytokines. Such assays are also well known in the art. Forexample, increased proliferation of cells can be demonstrated byincreased ³H-thymidine incorporation (due to increased DNA synthesisneeded for cellular mulitplication) or ELISA and/or RIA for detectingincreased production of cytokines by T cells in culture.

In one such experiment, PD-1 binding activity of human B7-DC-Ig wasassessed by ELISA. 96-well ELISA plates were coated with 100 uL 0.75ug/mL recombinant human PD-1/Fc (R&D Systems) diluted in BupHCarbonate/Bicarbonate pH 9.4 buffer (Pierce) for 2 hours and thenblocked with BSA solution (Jackson ImmunoResearch) for 90-120 minutes.Serially diluted human B7-DC-Ig (wild type, as well as D111S mutein, andK113S mutants that were selected for reduced binding to PD-1) as well ashuman IgG1 isotype control were allowed to bind for 90 minutes. BoundB7-DC-Ig was detected using 100 uL of 0.5 ug/mL biotin conjugatedanti-human B7-DC clone MIH18 (eBioscience) followed by 1:1000 dilutedHRP-Streptavidin (BD Bioscience) and TMB substrate (BioFX). Absorbanceat 450 nm was read using a plate reader (Molecular Devices) and datawere analyzed in SoftMax using a 4-parameter logistic fit. The datashowed that human B7-DC-Ig (wildtype) bound to PD-1 but the K113S andD111S mutants do not bind to PD-1.

In carrying out the procedures of the present invention it is of courseto be understood that reference to particular buffers, media, reagents,cells, culture conditions and the like are not intended to be limiting,but are to be read so as to include all related materials that one ofordinary skill in the art would recognize as being of interest or valuein the particular context in which that discussion is presented. Forexample, it is often possible to substitute one buffer system or culturemedium for another and still achieve similar, if not identical, results.Those of skill in the art will have sufficient knowledge of such systemsand methodologies so as to be able, without undue experimentation, tomake such substitutions as will optimally serve their purposes in usingthe methods and procedures disclosed herein.

The invention is described in more detail in the following non-limitingexamples. It is to be understood that these methods and examples in noway limit the invention to the embodiments described herein and thatother embodiments and uses will no doubt suggest themselves to thoseskilled in the art.

EXAMPLES Example 1 B7-DC-Ig binds to PD01 expressing CHO cells

B7-DC-Ig was first conjugated with allophycocyanin (APC) and thenincubated at various concentrations with a CHO cell line constitutivelyexpressing PD-1 or parent CHO cells that do not express PD-1. Bindingwas analyzed by flow cytometry. FIG. 1 shows the median fluorescenceintensity (MFI) of B7-DC-Ig-APC as a function of the concentration ofprobe (x-axis). B7-DC-Ig-APC binds to CHO.PD-1 cells (solid circle) butnot untransfected CHO cells (gray triangle).

Example 2 B7-DC-Ig competes with B7-H1 for binding to PD-1

B7-H1-Ig was first conjugated with allophycocyanin (APC). UnlabeledB7-DC-Ig at various concentrations was first incubated with a CHO cellline constitutively expressing PD-1 before adding B7-H1-Ig-APC to theprobe and cell mixture. FIG. 2 shows the median fluorescence intensity(MFI) of B7-H1-Fc-APC is shown as a function of the concentration ofunlabeled B7-DC-Ig competitor (x-axis) added. As the concentration ofunlabeled B7-DC-Ig is increased the amount of B7-H1-Ig-APC bound to CHOcells decreases, demonstrating that B7-DC-Ig competes with B7-H1 forbinding to PD-1.

Example 3 CT26 Tumor Model

-   -   Mouse colorectal tumor cell line, CT26, was obtained from ATCC.        A master cell bank at Passage 4 was generated following ATCC        guidelines. Cells were tested and confirmed no mycoplasma and        other pathogen contamination. One vial of tumor cells was thawed        from the cryopreserved stocks and grown for two passages prior        to inoculation.    -   CT26 cells were split at 1:5 dilution with 30 mL complete medium        (RPMI+10% FBS, 2 mM L-Glu, and 1× P/S) for two days culture or        at 1:10 dilution with 30 ml complete medium for 3 days culture.        CT26 cells were harvested by aspirating medium, rinsing the        flask with 5 mL PBS, adding 5 mL trypsin, incubating at 37° C.        for 2 min, and then neutralizing with 10 mL complete medium.        After centrifuge at 600×g (˜1000 rpm) for 5 min, media was        aspirated and the cell pellet was resuspended by pipetting with        10 ml plain RPMI. This wash step was repeated for three times.    -   Cell number and viability of the inoculated cells were analyzed        by trypan blue dye staining with proper dilution (e.g. 1:5        dilution, 10 μL cells+40 μL trypan blue) and confirmed by NOVA        cell count during the last wash step. Cell viability generally        was greater than 95% for inoculation.    -   CT26 cells were diluted to 6.7×10⁵ cells/mL for initial        inoculation with plain RPMI and stored on ice. Typically each        mouse was inoculated with 150 μL (1×10⁵ cells).    -   On Day 9, all the tumor-bearing mice were first grouped into a        rat cage and randomly divided the mice to experimental groups.        CTX solution was reconstituted by 1× PBS to 4 mg/mL. Mice were        intraperitoneally (IP) injected with 0.5 mL of CTX solution        resulting in 2 mg for a 20 gram mouse, i.e. 100 mg/kg.    -   On Day 10, mice were IP injected with 0.5 mL of B7-DC-Ig (0.2        mg/mL) resulting in 0.1 mg for a 20 gram mouse, i.e. 5 mg/kg.        The same dose was given 2 time a week for 4 weeks, total 8        doses. Tumor growth were monitored by measuring the tumor twice        weekly, starting on the day when giving B7-DC-Ig via a digital        caliper. Tumor volume was calculated as following:        Tumor volume=π(D _(short))²×(D _(long))/6=˜0.52×(D _(short))²×(D        _(long))    -   Mice were euthanized and taken off the study if the tumor volume        reached 2000 mm³ or if there were skin ulcers and infections at        the tumor inoculation site.

Example 4 Combination of Cyclophosphamide and B7-DC-Ig can EradicateEstablished Tumors

Balb/C mice at age of 9 to 11 weeks were implanted subcutaneously with1.0×10⁵ CT26 colorectal tumor cells as described above. On day 10 posttumor implantation, mice received 100 mg/kg of cyclophosphamide.B7-DC-Ig treatment started 1 day later, on day 11. Mice were treatedwith 100 ug of B7-DC-Ig, 2 doses per week, for 4 weeks and total 8doses. 75% of the mice that received the CTX+B7-DC-Ig treatment regimeneradicated the established tumors by Day 44, whereas all mice in thecontrol CTX alone group died as a result of tumor growth or wereeuthanized because tumors exceeded the sizes approved by IACUC (resultsshown in FIG. 3). These results demonstrate the effectiveness of thetreatment regimen on established tumors and not mere prophylaxis.

Example 5 Combination of Cyclophosphamide and B7-DC-Ig can EradicateEstablished Tumors and Protect Against Tumor Re-Challenge

Mice eradicated established CT26 colorectal tumors from the abovedescribed experiment were rechallenged with 1×10⁵ CT26 cells on Day 44and Day 70. No tumors grew out from the rechallenge suggesting they haddeveloped long term anti-tumor immunity from the cyclophosphamide andB7-DC-Ig combination treatment. All mice in the vehicle control groupdeveloped tumors (results shown in FIG. 4). These results show theeffectiveness of the treatment regimen on established tumors and thatthe cyclophosphamide and B7-DCIg combination treatment resulted inmemory responses to tumor antigens.

Example 6 Combination of Cyclophosphamide and B7-DC-Ig can GenerateTumor Specific, Memory Cytotoxic T Lymphocytes

Mice eradiated established CT26 colorectal tumors from the abovedescribed experiment were rechallenged with 2.5×10⁵ CT26 cells on Day44. Seven days later, mouse spleens were isolated. Mouse splenocyteswere pulsed with 5 or 50 ug/mL of ovalbumin (OVA) or AH1 peptides for 6hours in the presence of a Golgi blocker (BD BioScience). Memory Teffector cells were analyzed by assessing CD8⁺/IFNγ⁺ T cells. Results inFIG. 5 show that there were significant amount of CT26 specific Teffector cells in the CT26 tumor-eradicated mice.

Example 7 Effect of B7-DC-Ig Dose Dependent on Tumor Eradication

Balb/C mice at age of 9 to 11 weeks were implanted subcutaneously with1.0×10⁵ CT26 colorectal tumor cells. On day 9 post tumor implantation,mice received a single dose of cyclophosphamide (100 mg/kg) and startedtreatment on Day 10 with 30, 100 or 300 μg of B7-DC-Ig, 2 doses per weekfor 4 weeks, total 8 doses. FIG. 6 shows there were 70% of the miceeradicated the tumors at 300 μg, 40% tumor eradication with 100 μg, and30 μg dose gave rise to 10% tumor eradication.

Example 8 Combination of Cyclophosphamide and Anti-PD-1 can EradicateEstablished Tumors

Balb/C mice at age of 9 to 11 weeks were challenged subcutaneously with1.0×10⁵ CT26 colorectal tumor cells. On day 11 post tumor challenge,mice received a single dose of cyclophosphamide (100 mg/kg) and startedtreatment with anti-PD-1 antibody (250 ug, Clone G4, Hirano F. et al.,2005 Cancer Research) which was administered 3 times per week for fourweeks. 70% of the mice that received the CTX+ anti-PD-1 regimeneradicated established CT26 tumors at day 50 after tumor challenge,whereas all mice in the control and anti-PD-1 alone groups died as aresult of tumor growth or were euthanized because tumors exceeded thesizes approved by IACUC. These results show the effectiveness of thetreatment regimen on established tumors and not mere prophylaxis.Results are shown in FIG. 7.

Example 9 Combination of Cyclophosphamide and Anti-CTLA4 can EradicateEstablished Tumors

Balb/C mice at age of 9 to 11 weeks were challenged subcutaneously with1.0×10⁵ CT26 colorectal tumor cells. On day 11 post tumor challenge,mice received 100 mg/kg of cyclophosphamide. Anti-CTLA4 (an anti-mouseCTLA4 from hamster hybridoma—ATCC deposit UC10-4F10-11) treatment wasstarted 1 day later, on day 12. Mice were treated with 100 ug ofanti-CTLA4, 2 doses per week, for 4 weeks. 56% of the mice that receivedthe CTX+ anti-CTLA4 regimen were tumor free at day 50 after tumorchallenge, whereas all mice in the control group died as a result oftumor growth or were euthanized because tumors exceeded the sizesapproved by IACUC. Results are shown in FIG. 8. These results show theeffectiveness of the treatment regimen on established tumors and notmere prophylaxis.

Example 10 Combination of Cyclophosphamide and B7-DC-Ig Regimen Leads toReduction of Tregs in the Tumor Microenvironment

FIG. 9 shows the results of experiments wherein Balb/C mice at age of 9to 11 weeks of age were implanted with 1×10⁵ CT26 cells subcutaneously.On Day 9, mice were injected with 100 mg/kg of CTX, IP. Twenty fourhours later, on Day 10, mice were treated with 100 ug of B7-DC-Ig. Therewere 5 groups: naïve mice that did not receive any tumor cells, vehicleinjected, CTX alone, CTX+B7-DC-Ig or B7-DC-Ig alone. Two naïve mice and4 mice from other groups were removed from the study on Day 11 (2 dayspost CTX) and Day 16 (7 days post CTX) for T cell analysis. Left panelshows on Day 11, 2 days post CTX injection, Treg in the spleen of themice with CTX treatment was significantly lower than the one in the micewith tumor implantation and injected with vehicle. Right panel showsthat on Day 16, 7 days post CTX and 6 days post B7-DC-Ig treatment,B7-DC-Ig significantly lowered the CD4+ T cells expressing high PD-1.This was observed in both the B7-DC-Ig treated and CTX+B7-DC-Ig treatedmice. Mice implanted with tumor cells intended to have more PD-1+/CD4+ Tcells in the draining LN compared with naïve mice.

Example 11 Combination of Cyclophosphamide and B7-DC-Ig can PromoteMouse Survival in a Metastatic Prostate Tumor Model

B10.D2 mice at age of 9 to 11 weeks were injected intravenously with3.0×10⁵ SP-1 cells, which were isolated from lung metastasis post parentTRAMP cell injection. The CTX mice received 3 doses of CTX, 50 mg/kg, onDay 5, 12 and 19. The B7-DC-Ig treated mice received 3 doses ofB7-DC-Ig, 5 mg/kg, on Day 6, 13 and 20. On Day 100, 17% of mice in thecontrol groups, no-treated, CTX alone, B7-DC-Ig alone survived while 43%of the mice received combination of CTX and B7-DC-Ig survived. Resultsare shown in FIG. 10.

Example 12 Combination of Listeria Cancer Vaccine and B7-DC-Ig canEnhance Mouse Survival Post CT26 Liver Implantation

Balb/C mice at age of 11-13 weeks were implanted with CT26 cells using ahemispleen injection technique (Yoshimura K et al., 2007, CancerResearch). On Day 10, mice received 1 injection of CTX at 50 mg/kg, IP.Twenty four hours later, on Day 11, mice were treated with recombinantListeria carrying AH1 peptide, an immunodominant epitope of CT26, at 0.1LD₅₀ (1×10⁷ CFU), then on Day 14 and 17. Mice were also treated withB7-DC-Ig on Day 11 and then on Day 18. FIG. 11 shows mice without anytreatment or treated with CTX and Listeria cancer vaccine all diedbefore Day 45. There were 60% of the mice received triple combination,CTX+Listeria cancer vaccine and B7-DC-Ig survived.

1. Reference List

-   -   1. Brode S, Cooke A. Immune-potentiating effects of the        chemotherapeutic drug cyclophosphamide. Crit. Rev. Immunol.        2008; 28(2):109-26    -   2. van der Most R G, Currie A J, Mahendran S, Prosser A, Darabi        A, Robinson B W, Nowak A K, Lake R A. Tumor eradication after        cyclophosphamide depends on concurrent depletion of regulatory T        cells: a role for cycling TNFR2-expressing effector-suppressor T        cells in limiting effective chemotherapy. Cancer Immunol.        Immunother. 2009 August; 58(8):1219-28    -   3. Taieb J, Chaput N, Schartz N, Roux S, Novault S, Menard C,        Ghiringhelli F, Terme M, Carpentier A F, Darrasse-Jeze G, et al.        Chemoimmunotherapy of tumors: cyclophosphamide synergizes with        exosome based vaccines. J. Immunol. 2006 Mar. 1; 176(5):2722-9    -   4. Machiels J P, Reilly R T, Emens L A, Ercolini A M, Lei R Y,        Weintraub D, Okoye F I, Jaffee E M. Cyclophosphamide,        doxorubicin, and paclitaxel enhance the antitumor immune        response of granulocyte/macrophage-colony stimulating        factor-secreting whole-cell vaccines in HER-2/neu tolerized        mice. Cancer Res. 2001 May 1; 61(9):3689-97    -   5. Bass K K, Mastrangelo M J. Immunopotentiation with low-dose        cyclophosphamide in the active specific immunotherapy of cancer.        Cancer Immunol. Immunother. 1998 September; 47(1):1-12    -   6. Hengst J C, Mokyr M B, Dray S. Cooperation between        cyclophosphamide tumoricidal activity and host antitumor        immunity in the cure of mice bearing large MOPC-315 tumors.        Cancer Res. 1981 June; 41(6):2163-7    -   7. Hengst J C, Mokyr M B, Dray S. Importance of timing in        cyclophosphamide therapy of MOPC-315 tumor-bearing mice. Cancer        Res. 1980 July; 40(7):2135-41    -   8. Tsung K, Meko J B, Tsung Y L, Peplinski G R, Norton J A.        Immune response against large tumors eradicated by treatment        with cyclophosphamide and IL-12. J. Immunol. 1998 Feb. 1;        160(3):1369-77    -   9. Honeychurch J, Glennie M J, Illidge T M. Cyclophosphamide        inhibition of anti-CD40 monoclonal antibody-based therapy of B        cell lymphoma is dependent on CD11b+ cells. Cancer Res. 2005        Aug. 15; 65(16):7493-501    -   10. Wada S, Yoshimura K, Hipkiss E L, Harris T J, Yen H R,        Goldberg M V, Grosso J F, Getnet D, Demarzo A M, Netto G J,        Anders R, Pardoll D M, Drake C G. Cyclophosphamide augments        antitumor immunity: studies in an autochthonous prostate cancer        model. Cancer Res. 2009 May 15; 69(10):4309-18.    -   11. Freeman, G. J. Structures of PD-1 with its ligands: sideways        and dancing cheek to cheek. Proc. Natl. Acad. Sci. U.S. A 105,        10275-10276 (2008).    -   12. Brode, S. & Cooke, A. Immune-potentiating effects of the        chemotherapeutic drug cyclophosphamide. Crit. Rev. Immunol. 28,        109-126 (2008).    -   13. van der Most, R. G. et al. Tumor eradication after        cyclophosphamide depends on concurrent depletion of regulatory T        cells: a role for cycling TNFR2-expressing effector-suppressor T        cells in limiting effective chemotherapy. Cancer Immunol.        Immunother. 58, 1219-1228 (2009).    -   14. Taieb, J. et al. Chemoimmunotherapy of tumors:        cyclophosphamide synergizes with exosome based vaccines. J.        Immunol. 176, 2722-2729 (2006).    -   15. Bass, K. K. & Mastrangelo, M. J. Immunopotentiation with        low-dose cyclophosphamide in the active specific immunotherapy        of cancer. Cancer Immunol. Immunother. 47, 1-12 (1998).    -   16. Machiels, J. P. et al. Cyclophosphamide, doxorubicin, and        paclitaxel enhance the antitumor immune response of        granulocyte/macrophage-colony stimulating factor-secreting        whole-cell vaccines in HER-2/neu tolerized mice. Cancer Res. 61,        3689-3697 (2001).    -   17. Hengst, J. C., Mokyr, M. B., & Dray, S. Cooperation between        cyclophosphamide tumoricidal activity and host antitumor        immunity in the cure of mice bearing large MOPC-315 tumors.        Cancer Res. 41, 2163-2167 (1981).    -   18. Hengst, J. C., Mokyr, M. B., & Dray, S. Importance of timing        in cyclophosphamide therapy of MOPC-315 tumor-bearing mice.        Cancer Res. 40, 2135-2141 (1980).    -   19. Tsung, K., Meko, J. B., Tsung, Y. L., Peplinski, G. R., &        Norton, J. A. Immune response against large tumors eradicated by        treatment with cyclophosphamide and IL-12. J. Immunol. 160,        1369-1377 (1998).    -   20. Honeychurch, J., Glennie, M. J., & Illidge, T. M.        Cyclophosphamide inhibition of anti-CD40 monoclonal        antibody-based therapy of B cell lymphoma is dependent on CD11b+        cells. Cancer Res. 65, 7493-7501 (2005).    -   21. Wada S, Yoshimura K, Hipkiss E L, Harris T J, Yen H R,        Goldberg M V, Grosso J F, Getnet D, Demarzo A M, Netto G J,        Anders R, Pardoll D M, Drake C G. Cyclophosphamide augments        antitumor immunity: studies in an autochthonous prostate cancer        model. Cancer Res. 2009 May 15; 69(10):4309-18.

What is claimed is:
 1. A method of increasing a T cell response in ahuman comprising administering to the subject an anti-PD-1 antibody oran antigen binding fragment thereof in combination with a potentiatingagent selected from the group consisting of cyclophosphamide and ananalog of cyclophosphamide, wherein administration of the anti-PD-1antibody or antigen binding fragment thereof in combination with thepotentiating agent increases a T cell response, wherein the dose of thepotentiating agent is less than 200 mg/kg, wherein the dose of thepotentiating agent is not effective to have direct anti-tumor activity,and wherein the T cell response achieved by the combination of anti-PD-1antibody and the potentiating agent is greater than the T cell responseachieved by administering either the anti-PD-1 antibody alone or thepotentiating agent alone.
 2. The method of claim 1 wherein the anti-PD-1antibody or antigen binding fragment thereof inhibits, reduces,modulates or abolishes signal transduction mediated by PD-1.
 3. Themethod of claim 1 wherein the anti-PD-1 antibody is a human anti-PD-1antibody or antigen binding fragment thereof.
 4. The method of claim 1wherein the antigen binding fragment is a Fab, F(ab′)₂, or Fv fragment.5. The method of claim 1 wherein the anti-PD-1 antibody or antigenbinding fragment thereof is a humanized, chimeric, or single-chainantibody or fragment thereof.
 6. The method of claim 1 wherein thepotentiating agent reduces the activity of regulatory T lymphocytes(Tregs).
 7. The method of claim 1 wherein the amount of potentiatingagent is sub-therapeutic in the absence of the anti-PD-1 antibody or anantigen binding fragment thereof.
 8. The method of claim 1 wherein thedose of the potentiating agent is 8 mg/kg of cyclophosphamide or ananalog of cyclophosphamide.
 9. The method of claim 1 further comprisingadministering an additional immunomodulatory agent.
 10. The method ofclaim 1 further comprising administering an additional agent selectedfrom the group consisting of an anti-CTLA4 antibody, a mitosisinhibitor, an aromatase inhibitor, an A2a adenosine receptor (A2AR)antagonist, and an angiogenesis inhibitor.
 11. The method of claim 10wherein the angiogenesis inhibitor is a VEGF inhibitor.
 12. The methodof claim 11 wherein the VEGF inhibitor is bevacizumab or VEGF-Trap. 13.The method of claim 1 wherein the potentiating agent is administeredbefore the anti-PD-1 antibody or antigen binding fragment thereof. 14.The method of claim 13 wherein the potentiating agent is administered atleast 1, 2, 3, 5, 10, 15, 20, 24, or 30 hours before the anti-PD-1antibody or antigen binding fragment thereof.
 15. The method of claim 1wherein the anti-PD-1 antibody or antigen binding fragment thereof andthe potentiating agent are administered to a subject having cancer or aninfection.
 16. The method of claim 15 wherein the cancer is selectedfrom the group consisting of a bladder, brain, breast, cervical,colo-rectal, esophageal, kidney, liver, lung, nasopharangeal,pancreatic, prostate, skin, stomach, uterine, ovarian, testicular, andhematologic cancer.
 17. The method of claim 15 wherein the cancer isselected from the group consisting of gastric cancer, glioma, leukemia,melanoma, multiple myeloma, renal cell carcinoma, and urothelial cancer.18. The method of claim 15 wherein the cancer is melanoma.
 19. Themethod of claim 15 wherein the infection is a viral infection.
 20. Themethod of claim 19 wherein the viral infection is selected from thegroup consisting of, papilloma, herpes, encephalitis, influenza,hepatitis, and the common cold.
 21. The method of claim 19 wherein theviral infection is caused by a virus selected from the group consistingof, human papilloma virus (HPV), herpes simplex virus (HSV), humaninfluenza virus A, hepatitis C virus (HCV), hepatitis B virus (HBV), andhuman rhinovirus.
 22. The method of claim 15 wherein the infection is anon-viral infection.
 23. The method of claim 22 wherein the non-viralinfection is caused by a microorganism selected from the groupconsisting of Actinomyces, Anabaena, Bacillus, Bacteroides,Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter,Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium,Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium,Heliobacter, Haemophilus, Hemophilus influenza type B (HIB),Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A, Band C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma,Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus,Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella,Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces,Sulfolobus, Thermoplasma, Thiobacillus, Treponema, Vibrio, Yersinia,Cryptococcus neoformans, Histoplasma sp. (such as Histoplasmacapsulatum), Candida albicans, Candida tropicalis, Nocardia asteroides,Rickettsia ricketsii, Rickettsia typhi, Leishmania, Mycoplasmapneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium sp.(such as Plasmodium falciparum), Trypanosoma brucei, Entamoebahistolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosomamansoni.
 24. A pharmaceutical dosage unit comprising an anti-PD-1antibody or an antigen binding fragment thereof and a potentiating agentselected from the group consisting of cyclophosphamide and an analog ofcyclophosphamide, wherein administration of the dosage unit to a subjectincreases a T cell response in the subject, wherein the dose of thepotentiating agent is less than 200 mg/kg, wherein the dose ofpotentiating agent is not effective to have direct anti-tumor activity,and wherein the T cell response achieved by the dosage unit is greaterthan the T cell response achieved by administering either the anti-PD-1antibody alone or the potentiating agent alone.
 25. A method ofincreasing T cell response in a human comprising administering to ahuman the pharmaceutical dosage unit of claim
 24. 26. The pharmaceuticaldosage unit of claim 24 further comprising an antigen.
 27. The method ofclaim 15 wherein the T cell response against the cancer achieved by thecombination of the anti-PD1 antibody and the potentiating agent isgreater than the T cell response against the cancer achieved byadministering either the anti-PD 1 antibody alone or the potentiatingagent alone.
 28. The method of claim 1 wherein the dosage ofpotentiating agent is 5 mg/kg.